Abstract
The widespread TnpB proteins of IS200/IS605 transposon family have recently emerged as the smallest RNA-guided nucleases capable of targeted genome editing in eukaryotic cells1,2. Bioinformatic analysis identified TnpB proteins as the likely predecessors of Cas12 nucleases3,4,5, which along with Cas9 are widely used for targeted genome manipulation. Whereas Cas12 family nucleases are well characterized both biochemically and structurally6, the molecular mechanism of TnpB remains unknown. Here we present the cryogenic-electron microscopy structures of the Deinococcus radiodurans TnpB–reRNA (right-end transposon element-derived RNA) complex in DNA-bound and -free forms. The structures reveal the basic architecture of TnpB nuclease and the molecular mechanism for DNA target recognition and cleavage that is supported by biochemical experiments. Collectively, these results demonstrate that TnpB represents the minimal structural and functional core of the Cas12 protein family and provide a framework for developing TnpB-based genome editing tools.
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Data availability
All data are available in the paper and the supplementary material. The atomic coordinates and cryo-EM density maps have been deposited in the Protein Data Bank and Electron Microscopy Data Bank under accession codes 8BF8/EMD-16016 (binary complex), 8EXA/EMD-28656 (ternary conformation 1 complex) and 8EX9/EMD-28655 (ternary conformation 2 complex). Source data are provided with this paper.
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Acknowledgements
The research has been supported by the Novozyme Prize grant to V.S. and the Research Council of Lithuania grant no. S-MIP-21-8 to T.K. We thank the Danish Cryo-EM National Facility in CFIM at the University of Copenhagen and especially T. Pape and N. Sofos for support during cryo-EM data collection. We also thank I. Drulyte (Thermo Fisher Scientific) for invaluable help with cryo-EM sample preparation and data analysis, S. Erlendsson for preprocessing the initial data and G. Bigelyte for the help with plasmid construction. G.M. is part of the Novo Nordisk Foundation Center for Protein Research, which is supported financially by the Novo Nordisk Foundation (grant no. NNF14CC0001). This work was also supported by grant nos. NNF0024386 and NNF17SA0030214 and Distinguished Investigator grant no. NNF18OC0055061 to G.M., who is a member of the Integrative Structural Biology Cluster at the University of Copenhagen, and the Lundbeck Foundation (Lundbeckfonden) postdoctoral grant no. R380-2021-1448 to A.C.
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Contributions
G.S., G.T., G.D., A.C., G.M., T.K. and V.S. designed the research. G.D. and A.S. performed the protein purifications. G.S., G.T. and A.C. collected cryo-EM data. G.S. and G.T. prepared cryo-EM samples. G.S., G.T., A.C. and G.M. solved the structures. G.D. performed DNA binding and cleavage assays. G.S., G.T., D.K., Č.V. and T.K. performed computational sequence and structure analyses. G.S., G.T., T.K. and V.S. wrote the manuscript with input from all authors. All authors read and approved the final manuscript.
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T.K. and V.S. are co-inventors on a patent application (PCT/IB2021/055958) filed by Vilnius University. V.S. is a Chairman of and has financial interest in CasZyme. G.M. is a shareholder and a member of Ensoma SAB. The remaining authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Purification of ISDra2 TnpB RNP and design of DNA oligoduplexes.
a, Experimental workflow showing expression and multi-step purification of TnpB RNP complex. Addition of an HDV ribozyme sequence at the 3′-end ensured production of reRNA with a fixed 16-nt length guide sequence. b, SDS-PAGE analysis of the purified TnpB and TnpB (D191A) RNP complexes. Different amounts of purified protein samples were loaded on the gel. c, EMSA analysis of TnpB binding to the product-like partially single-stranded (left), substrate-like double-stranded (center) and nonspecific double-stranded (right) DNA oligoduplexes. d, Design of the head-to-head dual-end DNA oligoduplex that was used for structural analysis of TnpB-reRNA-DNA ternary complexes. e, EMSA analysis of TnpB binding to the dual-end DNA oligoduplex. Positions of complexes with one and two TnpB RNPs bound are indicated. f, Interpretation of cryo-EM data obtained with the dual-end DNA. During data processing, TnpB-reRNA-DNA complexes formed at each terminus of the dual-end DNA were interpreted as individual particles. The resultant cryo-EM reconstruction (bottom) in addition to the high quality map of the “primary complex” used for model building, also contained residual density corresponding to part of the second complex. Two copies of TnpB-reRNA-DNA complexes are shown fitted into both regions of the map. The angle between the long axes of these structures (blue lines) is 77°. Such fixed orientation of two TnpB RNPs bound to the dual-end DNA altered spatial distribution of the specimen, thereby alleviating the preferred orientation related problems. The sequences of the oligonucleotides are listed in Supplementary information Table 3. For uncropped gel images, see Supplementary information Fig. 1.
Extended Data Fig. 2 CryoEM single particle reconstruction of TnpB-reRNA binary and TnpB-reRNA-DNA ternary (conformation 1) complexes.
a, Workflow of the cryo-EM image processing and 3D reconstruction for the TnpB-reRNA binary complex. b, Workflow of the cryo-EM image processing and reconstruction for the TnpB-reRNA-DNA ternary conformation 1 (resolved RuvC domain) complex. The final electron density maps showing local resolution, masks from the local refinement jobs, directional distribution plots and FSC (Fourier shell correlation) plots are shown in black rectangles.
Extended Data Fig. 3 CryoEM single particle reconstruction of TnpB-reRNA-DNA ternary conformation 2 complex.
Two sets of data (untilted and tilted) were acquired and combined. The final electron density maps showing local resolution, mask from the local refinement job, directional distribution plots and FSC (Fourier shell correlation) plots are shown in a black rectangle.
Extended Data Fig. 4 Structural features of Deinococcus radiodurans ISDra2 TnpB ternary complexes.
a, Cryo-EM reconstruction (left) and cartoon representation (middle) of TnpB-reRNA-DNA ternary conformation 2 complex. Both sharpened (colored) and unsharpened (black outline) cryo-EM maps are shown. Bound nucleic acids and protein contacts in the complex are schematically depicted on the right. Dashed rectangular shapes encircle unresolved parts of the RNA/DNA. The dashed line on the cryo-EM map marks the boundary of the density corresponding to the primary (shown) and the secondary (cut-off) TnpB subunits, as explained in Extended Data Fig. 1f. b, Topology diagram of the ternary conformation 1 complex structure. Structural elements of the ZnF domains are based on an AlphaFold29 model. Helices and strands are shown as cylinders and arrows, respectively. Red circles mark the RuvC catalytic center residues D191, E278 and D361. c, Pairwise comparison of binary – ternary conformation 1, and ternary conformation 1 – ternary conformation 2 structures.
Extended Data Fig. 5 Effect of TAM mutations on binding and cleavage of dsDNA substrates.
Testing the TnpB RNP ability to bind (a and b, EMSA gels and their quantification plot, respectively), and cleave (c) dsDNA substrates, containing single nucleotide mutations in the TAM sequence. The dots in the line graph represent the mean of three independent reaction replicates (n = 3) ± standard deviation (s.d.). TAM and target sequences are highlighted in magenta and black, respectively. TS – target strand; NTS – non-target strand. P – cleavage products. D – catalytically inactive TnpB RNP complex (dTnpB – D191A). M – radiolabeled ssDNA ladder. For uncropped gel images, see Supplementary information Fig. 1.
Extended Data Fig. 6 TnpB-reRNA interactions with DNA.
a, Protein contacts to the first three base pairs of the RNA-DNA heteroduplex. b, The “lid” subdomain contacts. The “lid” subdomain sterically blocks the RuvC catalytic center and makes contacts to the C7:dG-7 and U8:dA-8 base pairs in RNA-DNA heteroduplex from the minor groove side. dsDNA oligonucleotide binding (c) and plasmid cleavage (d) by TnpB complex containing unmodified reRNA (TnpB-reRNA) and reRNA with altered targeting sequence (TnpB-reRNA_AG). Schematic representation of the reRNA and target sequences are provided at the top of the (c) panel. The sequences of the plasmids and oligonucleotides are listed in Supplementary information Table 1 and Supplementary information Table 3, respectively. For uncropped gel images, see Supplementary information Fig. 1.
Extended Data Fig. 7 Effect of single nucleotide mismatches on binding and cleavage of dsDNA substrates.
Testing the TnpB RNP ability to bind (a), and cleave (b) dsDNA substrates, containing single nucleotide mismatches at various positions (the number following “MM”) in the target sequence. The cleavage reactions were quenched at 0, 5, 15, and 60 min time points. TAM and target sequences are highlighted in magenta and black, respectively. NTS – non-target strand, P – cleavage products, Target – dsDNA substrate without mismatches, M – radiolabeled ssDNA marker. For uncropped gel images, see Supplementary information Fig. 1.
Extended Data Fig. 8 Structural and biochemical features of TnpB reRNA.
a, Schematic representation of unmodified (reRNA, left) and truncated (reRNA_trunc, right) reRNAs. The tetraloop introduced in truncated reRNA is shown in blue. b, Plasmid DNA cleavage by TnpB complex containing unmodified reRNA (TnpB-reRNA) and reRNA with truncated stem 1 (TnpB-reRNA_trunc). c, FQ reporter cleavage by TnpB-reRNA and TnpB-reRNA_trunc variants. The dots represent the mean of three independent reaction replicates (n = 3) ± standard deviation (s.d.). d, Comparison of RNA-DNA bound to ISDra2 TnpB and UnCas12f. Note that in TnpB ternary complex stem 1 is parallel to the RNA-DNA heteroduplex, but its UnCas12f (PDB ID: 7C7L)14 equivalent stem 4 is perpendicular to RNA-DNA, and position of TnpB stem 1 is occupied by the RuvC domain of the 2nd protein subunit. For uncropped gel images, see Supplementary information Fig. 1.
Extended Data Fig. 9 Conserved motifs in Cas12/TnpB RuvC domain.
Cas12/TnpB can be grouped into two supergroups ((Q/D)RD and (N/H)AD) according to the conserved residues (shown in a gray rectangle) in their RuvC-III motif. ISDra2 TnpB RuvC catalytic center residues D191, E278 and D361 are marked with red rectangles.
Supplementary information
Supplementary Information
Supplementary Figs. 1 (uncropped gel images for the Extended Data figures) and 2 (full graphs for collateral FQ-reporter trans-cleavage assay) and Supplementary Tables 1–3.
Supplementary Data
This file contains source data for Supplementary Fig. 2.
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Sasnauskas, G., Tamulaitiene, G., Druteika, G. et al. TnpB structure reveals minimal functional core of Cas12 nuclease family. Nature 616, 384–389 (2023). https://doi.org/10.1038/s41586-023-05826-x
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DOI: https://doi.org/10.1038/s41586-023-05826-x
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