Bacteria use adaptive immune systems encoded by CRISPR and Cas genes to maintain genomic integrity when challenged by pathogens and mobile genetic elements1,2,3. Type I CRISPR–Cas systems typically target foreign DNA for degradation via joint action of the ribonucleoprotein complex Cascade and the helicase–nuclease Cas34,5, but nuclease-deficient type I systems lacking Cas3 have been repurposed for RNA-guided transposition by bacterial Tn7-like transposons6,7. How CRISPR- and transposon-associated machineries collaborate during DNA targeting and insertion remains unknown. Here we describe structures of a TniQ–Cascade complex encoded by the Vibrio cholerae Tn6677 transposon using cryo-electron microscopy, revealing the mechanistic basis of this functional coupling. The cryo-electron microscopy maps enabled de novo modelling and refinement of the transposition protein TniQ, which binds to the Cascade complex as a dimer in a head-to-tail configuration, at the interface formed by Cas6 and Cas7 near the 3′ end of the CRISPR RNA (crRNA). The natural Cas8–Cas5 fusion protein binds the 5′ crRNA handle and contacts the TniQ dimer via a flexible insertion domain. A target DNA-bound structure reveals critical interactions necessary for protospacer-adjacent motif recognition and R-loop formation. This work lays the foundation for a structural understanding of how DNA targeting by TniQ–Cascade leads to downstream recruitment of additional transposase proteins, and will guide protein engineering efforts to leverage this system for programmable DNA insertions in genome-engineering applications.
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Dy, R. L., Richter, C., Salmond, G. P. C. & Fineran, P. C. Remarkable mechanisms in microbes to resist phage infections. Annu. Rev. Virol. 1, 307–331 (2014).
Hille, F. et al. The biology of CRISPR–Cas: backward and forward. Cell 172, 1239–1259 (2018).
Doron, S. et al. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359, eaar4120 (2018).
Sinkunas, T. et al. In vitro reconstitution of Cascade-mediated CRISPR immunity in Streptococcus thermophilus. EMBO J. 32, 385–394 (2013).
Redding, S. et al. Surveillance and processing of foreign DNA by the Escherichia coli CRISPR–Cas system. Cell 163, 854–865 (2015).
Peters, J. E., Makarova, K. S., Shmakov, S. & Koonin, E. V. Recruitment of CRISPR–Cas systems by Tn7-like transposons. Proc. Natl Acad. Sci. USA 114, E7358–E7366 (2017).
Klompe, S. E., Vo, P. L. H., Halpin-Healy, T. S. & Sternberg, S. H. Transposon-encoded CRISPR–Cas systems direct RNA-guided DNA integration. Nature 571, 219–225 (2019).
Peters, J. E. Tn7. Microbiol. Spectr. 2, https://doi.org/10.1128/microbiolspec.MDNA3-0010-2014 (2014).
Jackson, R. N. et al. Crystal structure of the CRISPR RNA-guided surveillance complex from Escherichia coli. Science 345, 1473–1479 (2014).
Chowdhury, S. et al. Structure reveals mechanisms of viral suppressors that intercept a CRISPR RNA-guided surveillance complex. Cell 169, 47–57 (2017).
Guo, T. W. et al. Cryo-EM structures reveal mechanism and inhibition of DNA targeting by a CRISPR–Cas surveillance complex. Cell 171, 414–426 (2017).
Mulepati, S., Héroux, A. & Bailey, S. Crystal structure of a CRISPR RNA-guided surveillance complex bound to a ssDNA target. Science 345, 1479–1484 (2014).
Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, 163 (2018).
Holder, J. W. & Craig, N. L. Architecture of the Tn7 posttransposition complex: an elaborate nucleoprotein structure. J. Mol. Biol. 401, 167–181 (2010).
Holm, L. & Laakso, L. M. Dali server update. Nucleic Acids Res. 44 (W1), W351–W355 (2016).
Aravind, L., Anantharaman, V., Balaji, S., Babu, M. M. & Iyer, L. M. The many faces of the helix–turn–helix domain: transcription regulation and beyond. FEMS Microbiol. Rev. 29, 231–262 (2005).
Krissinel, E. Stock-based detection of protein oligomeric states in jsPISA. Nucleic Acids Res. 43 (W1), W314–W319 (2015).
Xiao, Y., Luo, M., Dolan, A. E., Liao, M. & Ke, A. Structure basis for RNA-guided DNA degradation by Cascade and Cas3. Science 361, eaat0839 (2018).
Choi, K. Y., Spencer, J. M. & Craig, N. L. The Tn7 transposition regulator TnsC interacts with the transposase subunit TnsB and target selector TnsD. Proc. Natl Acad. Sci. USA 111, E2858–E2865 (2014).
Faure, G. et al. CRISPR–Cas in mobile genetic elements: counter-defence and beyond. Nat. Rev. Microbiol. 17, 513–525 (2019).
Strecker, J. et al. RNA-guided DNA insertion with CRISPR-associated transposases. Science 365, 48–53 (2019).
Zhang, K. Gctf: Real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).
Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).
Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D 74, 531–544 (2018).
Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997).
Nicholls, R. A., Fischer, M., McNicholas, S. & Murshudov, G. N. Conformation-independent structural comparison of macromolecules with ProSMART. Acta Crystallogr. D 70, 2487–2499 (2014).
Brown, A. et al. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr. D 71, 136–153 (2015).
Fernández, I. S., Bai, X.-C., Murshudov, G., Scheres, S. H. W. & Ramakrishnan, V. Initiation of translation by cricket paralysis virus IRES requires its translocation in the ribosome. Cell 157, 823–831 (2014).
Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).
Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).
We thank B. Grassucci and Z. Zhang for technical assistance with cryo-EM data acquisition. Part of this work was performed at the Simons Electron Microscopy Center and National Resource for Automated Molecular Microscopy located at the New York Structural Biology Center, supported by grants from the Simons Foundation (SF349247), NYSTAR and the NIH National Institute of General Medical Sciences (GM103310).
Columbia University has filed a patent application related to this work. S.E.K. and S.H.S. are inventors on other patents and patent applications related to CRISPR–Cas systems and uses thereof. S.H.S. is a co-founder and scientific advisor to Dahlia Biosciences and an equity holder in Dahlia Biosciences and Caribou Biosciences.
Peer review information Nature thanks Scott Bailey, Ronald Chalmers, David Taylor and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Representative negatively stained micrograph for 500 nM TniQ–Cascade. b, Left, representative cryo-EM image for 2 µM TniQ–Cascade. A small dataset of 200 images was collected in a Tecnai-F20 microscope equipped with a Gatan K2 camera. Right, reference-free two-dimensional class averages for this initial cryo-EM dataset. c, Left, representative image from a large dataset collected in a Tecnai Polara microscope equipped with a Gatan K3 detector. Middle, detailed two-dimensional class averages were obtained that were used for initial model generation using the SGD algorithm23 implemented in Relion313 (right). d, Image processing workflow used to identify the two main classes of the TniQ–cascade complex in open and closed conformations. Local refinements with soft masks were used to improve the quality of the map within the terminal protuberances of the complex. These maps were instrumental for de novo modelling and initial model refinement.
Extended Data Fig. 2 Fourier shell correlation curves, local resolution, and unsharpened filter maps for the TniQ–Cascade complex in closed conformation.
a, Gold-standard Fourier shell correlation (FSC) curve using half maps; the global resolution estimation is 3.4 Å by the FSC 0.143 criterion. b, Cross-validation model-versus-map FSC. Blue curve, FSC between the shacked model refined against half map 1; red curve, FSC against half map 2, not included in the refinement; black curve, FSC between final model against the final map. The overlap observed between the blue and red curves guarantees a non-overfitted model. c, Unsharpened map coloured according to local resolutions, as reported by RESMAP33. d, Final model coloured according to B-factors calculated by REFMAC28. e, A flexible Cas8 domain encompassing residues 277–385 contacts the TniQ dimer at the other side of the crescent shape. Applying a Gaussian filter of increasing width to the unsharpened map allows for a better visualization of this flexible region.
The V. cholerae I-F variant TniQ–Cascade complex (left) was superposed with Pseudomonas aeruginosa I-F Cascade11 (also known as Csy complex; middle, PDB ID: 6B45) and E. coli I-E Cascade9 (right, PDB ID: 4TVX). Shown are alignments of the entire complex (top), the Cas8 and Cas5 subunits with the 5′ crRNA handle (second from top), the Cas7 subunit with a fragment of crRNA (second from bottom) and the Cas6 subunit with the 3′ crRNA handle (bottom).
Extended Data Fig. 4 Representative cryo-EM densities for all the components of the TniQ–Cascade complex in closed conformation.
a, Final refined model of TniQ–Cascade, with Cas8 in purple, Cas7 monomers in green, Cas6 in salmon, the TniQ monomers in blue and yellow, and the crRNA in grey. b–h, Final refined model inserted in the final cryo-EM density for select regions of all the molecular components of the TniQ–Cascade complex. Residues are numbered.
a, Refined model for the TniQ–Cascade shown as ribbons inserted in the semi-transparent Van der Waals surface, coloured as in Fig. 1. b, c, Magnified view of Cas8, which interacts with the 5′ end of the crRNA. The inset shows electron density for the highlighted region, where the base of nucleotide C1 is stabilized by stacking interactions with arginine residues R584 and R424. d, Cas6 interacts with the 3′ end of the crRNA ‘handle’ (nucleotides 45–60). e, An arginine-rich α-helix is deeply inserted within the major groove of the terminal stem–loop. This interaction is mediated by electrostatic interactions between basic residues of Cas6 and the negatively charged phosphate backbone of the crRNA. f, Cas6 (salmon) also interacts with Cas7.1 (green), establishing a β-sheet formed by β-strands contributed from both proteins.
a, TniQ–Cascade residues that interact with the crRNA are indicated. Approximate location for all protein components of the complex are also shown, as well as the position of each Cas7 finger. b, TniQ–Cascade residues that interact with crRNA and target DNA, shown as in a.
Extended Data Fig. 7 FSC curves, local resolution, and local refined maps for the TniQ–Cascade complex in open conformation.
a, Gold-standard FSC curve using half maps; the global resolution estimation is 3.5 Å by the FSC 0.143 criterion. b, Cross-validation model-versus-map FSC. Blue curve, FSC between shacked model refined against half map 1; red curve, FSC against half map 2, not included in the refinement; black curve, FSC between final model against the final map. The overlapping between the blue and red curves guarantees a non-overfitted model. c, Unsharpened map coloured according to local resolutions, as reported by RESMAP33. Right, slice through the map shown on the left. d, Local refinements with soft masks improved the maps in flexible regions. Shown is the region of the map corresponding to the TniQ dimer. Unsharpened maps coloured according to the local resolution estimations are shown before (left) and after (right) masked refinements. e, Final model for the TniQ dimer region, coloured according to the local B-factors calculated by REFMAC28.
Extended Data Fig. 8 FSC curves, local resolution, and unsharpened filter maps for the DNA-bound TniQ–Cascade complex complex.
a, Gold-standard FSC curve using half maps; the global resolution estimation is 2.9 Å by the FSC 0.143 criterion. b, Cross-validation model-versus-map FSC. Blue curve, FSC between the shacked model refined against half map 1; red curve, FSC against half map 2, not included in the refinement; black curve, FSC between final model against the final map. The overlap observed between the blue and red curves guarantees a non-overfitted model. c, Left, unsharpened map coloured according to local resolutions, as reported by RESMAP33. dsDNA is visible at the top right projecting outside of the complex. Right, final model coloured according to B-factors calculated by REFMAC28.
Extended Data Fig. 9 Alignment of DNA-bound TniQ–Cascade with structurally similar Cascade complexes.
The DNA-bound structure of V. cholerae I-F variant TniQ–Cascade complex (left) was superposed with DNA-bound structures of P. aeruginosa I-F Cascade11 (also known as Csy complex; middle, PDB ID: 6B44) and E. coli I-E Cascade9 (right, PDB ID: 5H9F). Shown are alignments of the entire complex (top), the Cas8 and Cas5 subunits with the 5′ crRNA handle and double-stranded PAM DNA (middle top), the Cas7 subunit with a fragment of crRNA (middle bottom), and the Cas6 subunit with the 3′ crRNA handle (bottom).
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Halpin-Healy, T.S., Klompe, S.E., Sternberg, S.H. et al. Structural basis of DNA targeting by a transposon-encoded CRISPR–Cas system. Nature 577, 271–274 (2020). https://doi.org/10.1038/s41586-019-1849-0