Canonical CRISPR–Cas systems provide adaptive immunity against mobile genetic elements1. However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion2,3,4,5,6,7. Type V-K CRISPR-associated transposons rely on the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ7, but the molecular mechanism of RNA-directed DNA transposition has remained elusive. Here we report cryo-electron microscopic structures of a Cas12k-guide RNA–target DNA complex and a DNA-bound, polymeric TnsC filament from the CRISPR-associated transposon system of the photosynthetic cyanobacterium Scytonema hofmanni. The Cas12k complex structure reveals an intricate guide RNA architecture and critical interactions mediating RNA-guided target DNA recognition. TnsC helical filament assembly is ATP-dependent and accompanied by structural remodelling of the bound DNA duplex. In vivo transposition assays corroborate key features of the structures, and biochemical experiments show that TniQ restricts TnsC polymerization, while TnsB interacts directly with TnsC filaments to trigger their disassembly upon ATP hydrolysis. Together, these results suggest that RNA-directed target selection by Cas12k primes TnsC polymerization and DNA remodelling, generating a recruitment platform for TnsB to catalyse site-specific transposon insertion. Insights from this work will inform the development of CRISPR-associated transposons as programmable site-specific gene insertion tools.
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Maps and atomic coordinates of the reported cryo-EM structures have been deposited in the Electron Microscopy Data Bank under accession codes EMD-13486 (S. hofmanni Cas12k–sgRNA–DNA complex) and EMD-13489 (Tns-DNA filament), and the Protein Data Bank with accession codes 7PLA (Cas12k–sgRNA–DNA complex) and 7PLH (Tns–DNA filament). Structure factors and atomic coordinates of the reported X-ray crystallographic structure of TniQ has been deposited in the Protein Data Bank with accession code 7OXD.
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We thank M. Sawicka, L. Loeff and S. Sorrentino (University of Zurich Center for Microscopy and Image Analysis) for assistance with cryo-EM sample preparation and data collection; the Cryo-Electron Microscopy Service Platform at EMBL Heidelberg for instrument access and F. Weis for assistance with data collection; R. Ciuffa for advice on helical reconstruction; M. Pacesa for help with cryo-EM data processing; F. Boneberg and A. Walter for technical assistance; B. Blattmann at the Protein Crystallization Center (University of Zurich) for assistance with crystallization screening; M. Wang, V. Olieric and T. Tomizaki at the Swiss Light Source (Paul Scherrer Institute, Villigen, Switzerland) for assistance with X-ray diffraction measurements; S. Kreutzer and the ETH Genome Engineering and Measurement Lab for assistance with ddPCR assays; G. Riddhough (Life Science Editors) for assistance with manuscript editing. This work was supported by Swiss National Science Foundation Project Grant 31003A_182567 and European Research Council (ERC) Consolidator Grant no. ERC-CoG-820152. I.Q. was supported by FEBS and EMBO (ALTF 296-2020) long-term postdoctoral fellowships. M.S. is a member of the Biomolecular Structure and Mechanism PhD Program of the Life Science Zurich Graduate School. M.J. is an International Research Scholar of the Howard Hughes Medical Institute, and Vallee Scholar of the Bert L. and N. Kuggie Vallee Foundation.
The authors declare no competing interests.
Peer review information Nature thanks Hiroshi Nishimasu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Top: Size exclusion chromatography analysis of the Cas12k-sgRNA-target DNA complex. Middle: SDS-PAGE analysis of fractions from a. Proteins were visualised by Coomassie blue staining. Bottom: denaturing PAGE analysis of fractions from a. Nucleic acids were stained with a fluorescent dye (Gel Red). b, Representative negative stain EM micrographs of the ShCas12k-sgRNA-dsDNA complex at 68,000x magnification (top) and 180,000x magnification (bottom). Experiment was repeated three times independently with similar results. For gel source data, see Supplementary Fig. 1.
a, Cryo-EM image processing workflow for the Cas12k-sgRNA-target DNA complex. Fourier Shell Correlations (FSC) of ShCas12k reconstruction from two independently refined half-maps. The gold-standard cutoff (FSC = 0.143) is marked with a blue line. b, Final electron density map colored according to the local resolution. c, Validation of Cas12k-sgRNA-target DNA structure model.
a, Close-up view of the RNA:DNA duplex in proximity of the bridge helix (BH). b, Close-up view of the sgRNA-TS DNA heteroduplex in the Cas12k-sgRNA-target DNA complex. c, Structural model of the Cas12k sgRNA with detailed views of the 5′ terminal segment (top left, 6.6 σ contour level) and the triplex junction (bottom right, 9.0 σ contour level) of the tracrRNA forming ribose-zipper and A-minor interactions, the pseudoknot duplex (bottom left, 9.0 σ contour level) and the central stack junction (top right, 7.6 σ contour level). d, Droplet digital PCR (ddPCR)-based analysis of the transposition activity of structure-based sgRNA scaffold mutants in the ShCAST system. Data are presented as mean ± s.d. (n=3 biologically independent replicates).
Extended Data Fig. 5 ShTnsC filament formation and cryo-EM image processing of TnsC-dsDNA helical filaments.
a, Representative negative-stain EM micrographs of TnsC in the presence of a 92-bp dsDNA and ATP or ATPγS. Scale bars, 100 nm. Magnification, 120,000x. Experiment was repeated twice independently with similar results. b, Cryo-EM data processing workflow for the TnsC-dsDNA complex. Selected 2D class averages used in image reconstruction and intermediate and final reconstructions are shown. c, Fourier Shell Correlations (FSC) of TnsC filament reconstruction from two independently refined half-maps. The gold-standard cutoff (FSC = 0.143) is marked with dashed lines. The resolution value of the FSC corrected curve at this level is indicated. d, Structural alignment of the cryo-EM structure of S. hofmanni TnsC (blue) and the crystal structure of the Aeropyrum pernix ORC2 protein (grey; PDB ID: 1W5T52) used as initial homology model for model building (single protomers). The root mean square deviation (RMSD) between the structures is 1.41 Å over 79 pruned atom pairs, as calculated in Chimera. e, Local resolution estimation (Å) for the cryo-EM volumes of the TnsC-dsDNA helical filaments. f, Local resolution estimation (Å) of the bound DNA duplex.
a, SDS-PAGE analysis of purified wild type and ATPase activity mutant TnsC proteins. b, Representative negative-stain EM micrographs of TnsC ATPase activity mutants incubated in the presence of dsDNA and ATP or AMPPNP. Magnification, 98,000x. Experiment was repeated twice independently with similar results. c, SDS-PAGE analysis of purified wild type and DNA binding mutant TnsC proteins. d, Representative negative stain EM micrographs of TnsC in the presence of AMPPNP and dsDNA and with mutations in the DNA binding interface. Magnification, 98,000x. Experiment was repeated twice independently with similar results. e, Electromobility shift assay using fluorophore-labeled 27-bp dsDNA in the presence of AMPPNP and either wild type or DNA binding mutant TnsC proteins. -, no protein control. Experiment was repeated twice independently with similar results. For gel source data, see Supplementary Fig. 1.
a, Representative negative stain EM micrographs of dsDNA-bound TnsC filaments in the presence of TnsB and AMPPNP or ATP. Magnification, 98,000x. b, Quantification of filament length in the indicated samples. Data represents the average length ± s.d. of 50 randomly selected filaments in each sample. Experiment in a and quantification in b were repeated twice independently with similar results. c, Top panel: Domain architecture of TniQ proteins. HTH, helix turn helix motif. wHTH, winged HTH. ZnF, zinc-finger motif. Bottom panel: Structural alignment of the crystal structures of type V S. hofmanni TniQ (red) and type I-F Vibrio cholerae TniQ (grey and beige protomers; PDB ID: 6V9P19) (bottom panel). The root mean square deviation (RMSD) between the structures is 1.16 Å over 55 pruned atom pairs, as calculated in Chimera. d, Size exclusion chromatography analysis of TniQ. The retention volume corresponds to that of a protein of 12 kDa, as calculated based on molecular weight standards. The theoretical molecular weight of S. hofmanni TniQ is 19 kDa.
Schematic diagram depicting transposition of type V-K CRISPR-associated transposons. Cas12k in association with a crRNA-tracrRNA dual guide RNA recognizes target DNA sequences, forming a partial R-loop structure. Full R-loop is formed upon recruitment of TnsC by interactions with DNA-bound Cas12k, which nucleates ATP-dependent formation of a helical filament around structurally remodeled DNA. Filament growth is restricted by TniQ capping the Cas12k-distal end of the TnsC filament. The TnsC filament serves as a recruitment platform for TnsB, which interacts directly with TnsC and stimulates its ATPase activity. This leads to filament disassembly, making DNA downstream of the PAM accessible for transposon insertion. In the post-hydrolysis state, TnsC forms a single-turn helical hexamer around target DNA, possibly acting as a molecular ruler to define the spacing between the Cas12k binding and transposon insertion sites (60-66 nt downstream of the PAM). Image created with BioRender.com and adapted.
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Querques, I., Schmitz, M., Oberli, S. et al. Target site selection and remodelling by type V CRISPR-transposon systems. Nature 599, 497–502 (2021). https://doi.org/10.1038/s41586-021-04030-z