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Structure reveals why genome folding is necessary for site-specific integration of foreign DNA into CRISPR arrays

Nature Structural & Molecular Biology volume 30, pages 1675–1685 (2023)Cite this article

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Abstract

Bacteria and archaea acquire resistance to viruses and plasmids by integrating fragments of foreign DNA into the first repeat of a CRISPR array. However, the mechanism of site-specific integration remains poorly understood. Here, we determine a 560-kDa integration complex structure that explains how Pseudomonas aeruginosa Cas (Cas1–Cas2/3) and non-Cas proteins (for example, integration host factor) fold 150 base pairs of host DNA into a U-shaped bend and a loop that protrude from Cas1–2/3 at right angles. The U-shaped bend traps foreign DNA on one face of the Cas1–2/3 integrase, while the loop places the first CRISPR repeat in the Cas1 active site. Both Cas3 proteins rotate 100 degrees to expose DNA-binding sites on either side of the Cas2 homodimer, which each bind an inverted repeat motif in the leader. Leader sequence motifs direct Cas1–2/3-mediated integration to diverse repeat sequences that have a 5′-GT. Collectively, this work reveals new DNA-binding surfaces on Cas2 that are critical for DNA folding and site-specific delivery of foreign DNA.

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Fig. 1: Cryo-EM structure of the type I-F CRISPR integration complex.
Fig. 2: Foreign DNA constrains the Cas2/3 linker against conserved Cas1 residues.
Fig. 3: The Cas2 homodimer simultaneously coordinates four dsDNA helices critical to CRISPR integration.
Fig. 4: Sequence motifs in the leader and IHF proteins facilitate Cas1–2/3-based integration into diverse repeat sequences.
Fig. 5: I-F CRISPR integration complex suggests a mechanism for primed acquisition and Cascade impact on integration.
Fig. 6: DNA is a flexible scaffold that controls DNA mobilization.

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Data availability

The data that support the findings of this study are available from the corresponding author upon request. Curated raw multi-frame movies have been deposited along with a reference gain file under accession number EMPIAR-11659. Cryo-EM maps were deposited in the Electron Microscopy Data Bank under accession number EMD-29280. The atomic model of the type I-F integration complex was deposited in the PDB under accession number 8FLJ. Plasmids generated in this study are available from Addgene. Source data are provided with this paper.

Code availability

Code is available at https://github.com/WiedenheftLab/.

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Acknowledgements

Thanks to members of the B.W. laboratory for feedback and discussions. We thank M. Matyszewski and J. Jeliazkov for helpful discussions. Thanks to C. Hophan-Nichols for computational support. A.S.-F. is a postdoctoral fellow of the Life Science Research Foundation that is supported by the Simons Foundation. A.S.-F. is supported by the Postdoctoral Enrichment Program Award from the Burroughs Wellcome Fund. This work was supported by National Institutes of Health, United States grant 1K99GM147842 (A.S.-F.). L.T. and A.B.G. are supported by Montana State University’s Undergraduate Scholars Program, and by the NIH NIGMS IDeA program (P20GM103474). This work was performed using the cryo-EM facility at Montana State University (NSF 1828765 and the M.J. Murdock Charitable Trust). Microscopy was also performed at the National Center for CryoEM Access and Training (NCCAT) and the Simons Electron Microscopy Center located at the New York Structural Biology Center, supported by the NIH Common Fund Transformative High Resolution Cryo-Electron Microscopy program (U24 GM129539), and by grants from the Simons Foundation (SF349247) and NY State Assembly. Research in the Wiedenheft laboratory is supported by the NIH (R35GM134867), the M.J. Murdock Charitable Trust, a young investigator award from Amgen, the Montana State University Agricultural Experimental Station (USDA NIFA) and a sponsored research agreement from VIRIS Detection Systems. Molecular graphics and analyses performed with UCSF ChimeraX, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases. Funders had no role in the conceptualization, designing, data collection, analysis, decision to publish or preparation of the manuscript.

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Authors and Affiliations

  1. Department of Microbiology and Cell Biology, Montana State University, Bozeman, MT, USA

    Andrew Santiago-Frangos, William S. Henriques, Tanner Wiegand, Murat Buyukyoruk, Ava B. Graham, Royce A. Wilkinson, Lenny Triem & Blake Wiedenheft

  2. Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT, USA

    Colin C. Gauvin

  3. Thermal Biology Institute, Montana State University, Bozeman, MT, USA

    Colin C. Gauvin

  4. Simons Electron Microscopy Center, National Resource for Automated Molecular Microscopy, New York Structural Biology Center, New York, NY, USA

    Kasahun Neselu & Edward T. Eng

  5. Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA

    Gabriel C. Lander

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Contributions

A.S.-F. carried out conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, supervision, validation, visualization and writing (original draft). W.S.H., T.W. and M.B. performed data curation, investigation, methodology, visualization and writing (review and editing). A.B.G. and R.A.W. undertook investigation and methodology. L.T. carried out visualization. C.C.G. contributed to software, resources and writing (review and editing). K.N. and E.T.E. performed investigation and resources. G.C.L. undertook methodology, supervision, visualization and writing (review and editing). B.W. was responsible for funding acquisition, project administration, resources, supervision, visualization and writing (review and editing).

Corresponding author

Correspondence to Blake Wiedenheft.

Ethics declarations

Competing interests

B.W. is the founder of SurGene and VIRIS Detection Systems. B.W. and A.S.-F. are inventors on patent applications related to CRISPR–Cas systems and applications thereof. The remaining authors declare no competing interests.

Peer review

Peer review information

Nature Structural & Molecular Biology thanks Elizabeth Kellogg for her contribution to the peer review of this work. Dimitris Typas was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. Peer reviewer reports are available.

Additional information

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

Extended data

Extended Data Fig. 1 Cryo-EM sample preparation, imaging and processing for type I-F integration complex.

a, Sequence-level schematic of DNA used to assemble the integration complex. The length of each motif is listed. The latter two thirds of the CRISPR repeat and second spacer (grey dashed box) could not be resolved in the cryo-EM reconstruction. See also Supplementary Table 1. b, Size-exclusion chromatography (SEC) profile (Superdex 75 16/600, Cytiva) of IHF heterodimer purified as described in methods section, and SDS-PAGE gel (inset). c, SEC profile (Superdex 200 10/300, Cytiva) of Cas1–2/3 heterohexamer purified as described in methods section, and SDS-PAGE gel (inset). d, I-F integration complex was assembled from purified DNAs, IHF and Cas1–2/3 as described in the methods section, and the assembled complex was further purified by size-exclusion chromatography (SEC) (Superdex 200 10/300, Cytiva). Individual fractions were collected along the elution profile, and were concentrated and stored separately for further analysis and imaging. e, Individual SEC fractions were analyzed by SDS-PAGE to determine which fractions contained all the proteins necessary for a complete complex. f, Individual SEC fractions were phenol-chloroform extracted, and the aqueous layer was analyzed by Urea-PAGE to determine which fractions contained all four DNA strands necessary for a complete complex. The fraction chosen for cryo-EM analysis is indicated with a dotted purple box. g, Image processing pipeline for a small subset of 10,740 total micrographs for the type I-F integration complex, to generate an initial model for template picking. Scale bar represents 100 nm. h, Final image processing pipeline for the type I-F integration complex. i, Viewing direction distribution plot depicting particle orientations present in final reconstruction. More populated views are shown in red, and less populated views are shown in blue. j, 3D Fourier Shell Correlation (3DFSC) of the final I-F integration complex reconstruction. The global resolution at 0.143 is indicated by a dashed line, 3.48 Å. k, Local resolution estimation of the cryo-EM reconstruction calculated by cryoSPARC81. The purification of proteins, assembly of the integration complex, and analysis of these samples by SDS-PAGE or Urea page was performed once. Micrographs were collected on two separate occasions with similar results.

Source data

Extended Data Fig. 2 Cas1-2/3 undergoes a large structural rearrangement during integration.

a, The Cas3 domains of the Cas1-2/3 complex have undergone a ~ 100° rotation in the structure of the integration complex as compared to Cas1-2/3 alone54. The positions of the first (90) and last residues (110) of the Cas2/3 linker are shown (Cas2/3a, red; Cas2/3b, tomato). The linker residues were not resolved for the previously determined pseudo-atomic model of Cas1-2/3 alone, and so are not shown for either complex for clarity54. The rotation of the Cas3 domains outwards unveils new DNA binding sites on two opposing faces of the Cas2 dimer. b, Zoom-in on the atomic fit of the Cas2/3 linker to the cryo-EM map. The Cas2/3 linker is disordered in the absence of Cas1 (PDB:5B7I)55, but has become ordered in the I-F integration complex structure due to packing by the foreign DNA against the Cas1 beta hairpins. c, A sequence logo depicting the conservation of Cas2/3 linker residues (top). The Cas1 residues that contact the Cas2/3 linker are conserved in Cas1 proteins associated with type I-F CRISPR loci (middle), but are not conserved in the closely related Cas1 proteins associated with type I-E CRISPR loci that have similar IHF motif-containing leaders22. Residues are numbered according to P. aeruginosa PA14 Cas1 and Cas2/3 proteins.

Extended Data Fig. 3 Conservation analysis of Cas1-2/3 residues involved in DNA binding and integration.

a, Conservation of Cas1 and Cas2/3 residues involved in binding the foreign DNA, or catalyzing the strand transfer reaction, or catalyzing the degradation of nucleic acids. See Fig. 2. b, Conservation of basic and polar Cas1 and Cas2/3 residues involved in accommodating the DNA duplexes bound by the Cas1-2/3 complex during integration. See Fig. 3. Residues are numbered according to P. aeruginosa Cas1 and Cas2/3 proteins.

Extended Data Fig. 4 Cas1-2/3 predominantly recognizes IR motifs, CRISPR repeat and foreign DNA through non-sequence specific interactions.

a, Splayed 3′ ends of the foreign DNA are directed into the Cas1 transesterification active site. The product of the first strand-transfer reaction is shown in the Cas1a* active site (top), and the 3′ OH of the other end of the foreign DNA is positioned in the Cas1b* active site (bottom). The cryo-EM map is shown in transparent grey. b, Zoom-in on the Cas1-2/3 contacts to the CRISPR repeat (ChimeraX contacts command with default parameters). Most protein contacts occur to the DNA backbone and minor groove. Cas1 residue E184 appears to probe nucleotide G1 of the repeat. c, Zoom-in on the Cas1-2/3 contacts to the IR leader motifs. Most protein contacts occur to the DNA backbone and minor groove. d, DNAproDB analysis of Cas1-2/3 interactions with the 3′ ends of foreign DNA and the CRISPR leader-repeat junction. For clarity, only protein interactions to the nucleobases are shown93. e, DNAproDB analysis of Cas1-2/3 interactions with the IR leader motifs93. For clarity, only protein interactions to the nucleobases are shown. f, Zoom-in on the atomic fit of the base-pairs around the leader-repeat junction (-3 and +3 bps, coordinated by Cas1a*), to the cryo-EM map. Tension in the DNA loop at the leader-repeat junction has been released in the post-integration structure by a physical separation of base-pairs, as measured by an increase in base step rise. This tension may further pull the leaving 3′OH out of the Cas1 transesterification active site, to inhibit disintegration of the foreign DNA from the repeat. The approximate local base step rise was calculated using the http://web.x3dna.org/ webserver. g, A bioinformatic analysis of the first repeat from 24,940 CRISPR loci reveals that a 5′ GT dinucleotide is strongly conserved across most CRISPR subtypes. Similarly, a 5′ GT is present at the spacer-end of the repeat (seen as AC-3′ on the sense strand) within certain CRISPR subtypes (I-D, II-C, III-C, III-D, V-B, V-E, V-K, VI-A), but it is not broadly conserved.

Extended Data Fig. 5 Purification of structure-guided mutants of Cas1-2/3.

a, SEC profile of a new preparation of wildtype Cas1-2/3 and all variants purified in the same manner on a Superdex 200 16/600 (Cytiva). An excess of free Strep-tagged Cas1 elutes at approximately 82 mL. b, SDS-PAGE gel of the Cas1-2/3 hetero-hexamer peak for all purified Cas1-2/3 variants. The SDS-PAGE gel of all Cas1-2/3 samples was run twice with similar results.

Source data

Extended Data Fig. 6 Validation of Cas1-2/3 interactions with the foreign DNA and IR motifs.

a, Time-course integration reactions to test the role of Cas1H25 in splaying the foreign DNA ends. Integration reactions were performed with trimmed foreign DNA (lacking a PAM) in triplicate, resolved on denaturing polyacrylamide gels. Timepoints were taken at 0, 1, 2, 4 and 8 minutes. Reactions were stopped by the addition of phenol. A 32P-labelled DNA that is shorter (140-160 bp) than the full length CRISPR is present in some DNA preparations (also see Extended Data Fig. 6c). Full-length CRISPR DNA, leader- and spacer-side integration products, do not overlap with this band. Further, Cas1-2/3, foreign DNA and IHF are in excess over the 32P-labelled DNA. The 140-160 bp band does not interfere with the quantification or generation of integration products. b, Quantification of time-course experiments to determine the role of Cas1 residue H25 in integration. The mean and standard deviation of three replicate experiments are shown. The Cas1H25A mutant integrates splayed and fully complementary foreign DNA fragments less efficiently that WT Cas1-2/3, suggesting that H25 steers the non-nucleophilic DNA strand away from the Cas1 active site. These results mirror the previously published effect of type I-E Cas1-2 tyrosine wedge mutation10. c, Time-course integration reactions to test the role of Cas2 residues in recognition of the IR motifs in the leader, performed as in panel a. d, Quantification of time-course experiments to determine the role of Cas2 residues K11, R12, R55 and N56 in integration. The mean and standard deviation of three replicate experiments are shown. The Cas2R55E,N56D/3 mutant retains a small amount of integration activity. The Cas2K11D,R12E/3 and Cas2K11D,R12E,R55E,N56D/3 mutants do not integrate DNA into the I-F CRISPR. Quantification of leader- (grey circles) or spacer-side (white circles) integration events from all three replicate gels. Individual dots for each triplicate reaction are shown, and some dots overlap.

Source data

Extended Data Fig. 7 PAM blocks Cas-mediated integration of foreign DNA into CRISPR repeat.

a, Schematic summarizing the step-wise strand-transfer reactions catalyzed by a Cas integrase. In the absence of putative host nucleases that trim PAMs, the foreign DNA fragments that contain a PAM stall the reaction at leader-side integration. However, the foreign DNA fragments that have been trimmed (no PAM) proceed through leader- and spacer-side integration. b-d, Endpoint integration reactions performed with a PAM-containing foreign DNA in triplicate, resolved on denaturing polyacrylamide gels. The X1 and X2 lanes signify lanes that were not further analyzed for this manuscript. e-g, Endpoint integration reactions performed with a trimmed foreign DNA in triplicate, resolved on denaturing polyacrylamide gels. The X1 and X2 lanes signify integration substrates that were not analyzed for this manuscript. h, i, Quantification of leader- (grey circles) or spacer-side (white circles) integration events from all three replicate gels. Individual dots for each triplicate reaction are shown, and some dots overlap. Three independent gels were run for PAM-containing or trimmed Foreign DNA integration reactions with similar results.

Source data

Extended Data Fig. 8 Control reactions for integration assay and generation of 32P-labelled ladder.

a, Scheme of nine CRISPR fragments used for in vitro integration assays. Each CRISPR locus contains two repeats and two spacers. Leader motifs are color-coded and annotated (IR, inverted repeat; DR, direct repeat; IHF, IHF binding site; LAS, Leader anchoring site). To simplify the pictograms shown here and in subsequent panels, a single-colored rectangle was used to represent a given collection of leader motifs, and a single diamond was used to represent a CRISPR locus composed of two repeats and two spacers. b, The four CRISPR repeats tested in the integration assays have diverse palindromes (yellow), and a wide range of GC-content. c, Control reactions in which all components necessary for integration, except Cas1-2/3, were incubated. The overexposed gels show that the majority of the 32P signal for a given integration substrate DNA corresponds to the full-length strands. d, A custom 32P-labelled DNA ladder was made by mixing the degradation products generated by individual restriction enzyme digests of different 32P-labelled integration substrate DNAs. X1 and X2 signify integration substrates that were not analyzed for this manuscript. e, Schematics of the nine CRISPRs tested in Extended Data Fig. 5. The arrows identify locations of off-target integration reactions. Most off-target integration reactions occur by spurious integration at DNA motifs (for example, second CRISPR repeat, IHFdistal site, or upstream motifs) found near the ends of the CRISPR DNA target. Previous deep-sequencing of similar integration reactions has shown that the second repeat is a common off-target integration site, and that IHF blocks integration at the IHF binding site22. Urea-PAGE gels were run once.

Source data

Extended Data Fig. 9 Validation of Cas1-2/3 interactions with the repeat.

a, Time-course integration reactions to compare rate of integration into I-E and I-F repeats downstream of a I-F leader. b, Quantification of time-course experiments to determine the impact of 19 mutations associated with swapping the I-F repeat for the I-E repeat, on integration. Leader-side integration is indistinguishable. But spacer-side integration is slower into the I-E repeat. The mean and standard deviation of three replicate experiments are shown. c, Time-course integration reactions to measure the impact of I-F repeat mutations on integration rate. d, Time-course integration reactions to measure the impact of I-F repeat mutations on integration rate, in the context of a Cas1E184A mutation. The Cas1E184A mutation is expected to disrupt 5′ G recognition, but also impacts stability of the Cas1-2/3 complex (Extended Data Fig. 5). e, Quantification of time-course experiments to determine the impact of I-F repeat mutations on integration rate, in the context of WT Cas1-2/3. The mean and standard deviation of three replicate experiments are shown. f, Quantification of time-course experiments to determine the impact of I-F repeat mutations on integration rate, in the context of Cas1E184A-2/3. In panels e and f, no integration occurs into either the ‘G1A,T2A,A28C’ or ‘G1A,T2A’ repeat mutations, and the datapoints for these plots overlaps at roughly Y = 0 over the time course. The mean and standard deviation of three replicate experiments are shown. Each Urea-PAGE gel was run once.

Source data

Supplementary information

Supplementary Information

Supplementary Table 1.

Reporting Summary

Peer Review File

Supplementary Video 1

Cas1–2/3 undergoes a large conformational change to unveil Cas2-leader binding sites.

Supplementary Video 2

Overview of how the I-F CRISPR leader and IHF guide Cas1–2/3-mediated integration of foreign DNA at the first CRISPR repeat.

Supplementary Video 3

The strained IHF-mediated DNA bends sway in relation to the Cas1–2/3 integrase.

Source data

Source Data Fig. 4

Unprocessed gel images.

Source Data Extended Data Fig. 1

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Source Data Extended Data Fig. 5

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Source Data Extended Data Fig. 6

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Source Data Extended Data Fig. 7

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Source Data Extended Data Fig. 8

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Source Data Extended Data Fig. 9

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Santiago-Frangos, A., Henriques, W.S., Wiegand, T. et al. Structure reveals why genome folding is necessary for site-specific integration of foreign DNA into CRISPR arrays. Nat Struct Mol Biol 30, 1675–1685 (2023). https://doi.org/10.1038/s41594-023-01097-2

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