Cryo-EM structure of the transposon-associated TnpB enzyme – Nature

Sample preparation

The genes encoding ISDra2 TnpB (TnpB from D. radiodurans ISDra2; residues 1 to 408) and the 247-nt ωRNA were synthesized by Eurofin Genomics and cloned into the modified pETDuet vector (Novagen). The N-terminally MBP-tagged TnpB and ωRNA were co-expressed in Escherichia coli Rosetta2 (DE3). The E. coli cells were cultured at 37 °C until the A₆₀₀ reached 0.8, and protein expression was then induced by the addition of 0.2 mM isopropyl β-d-thiogalactopyranoside (Nacalai Tesque). The E. coli cells were further cultured at 20 °C overnight, collected by centrifugation, resuspended in buffer A (20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 5% glycerol, and 1 mM DTT), and then lysed by sonication. The lysates were centrifuged, and the supernatant was mixed with 3 ml of amylose resin (New England Biolabs). The mixture was loaded into a Poly-Prep column (Bio-Rad), and the TnpB–ωRNA complex was eluted with buffer B (20 mM Tris-HCl, pH 8.0, 40 mM maltose, 0.2 M NaCl, 5% glycerol, and 1 mM DTT). The complex was incubated with HRV3C protease overnight, and then loaded onto a 5 ml HiTrap Heparin column (GE Healthcare) equilibrated with buffer C (20 mM Tris-HCl, pH 8.0, and 0.2 M NaCl). The peak fractions were collected and stored at −80 °C in buffer D (20 mM Tris-HCl, pH 8.0, 0.2 M NaCl and 20% glycerol) until use. Mutations were introduced by a PCR-based method, and sequences were confirmed by DNA sequencing (Supplementary Tables 1 and 2). Since the 3′ end of the tnpB gene overlapped with part of the ωRNA, it was difficult to perform PCR with the plasmid containing the full-length ωRNA. Thus, all TnpB mutants were created by introducing mutations on the DNA plasmid containing ωRNA with deleted 5′ region (−231 to −117).

Cryo-EM analysis

The TnpB–ωRNA–target DNA ternary complex was prepared for cryo-EM analysis according to the following procedure. A double-stranded DNA with phosphorothioate modifications at the cleavage sites was prepared by annealing a 35-nt target DNA strand and a 35-nt non-target strand containing a TTGAT TAM, at 95 °C for 2 min. The purified TnpB–ωRNA complex was incubated with the target DNA at room temperature for 30 min. The TnpB–ωRNA–target DNA ternary complex was purified on a Superdex 200 Increase 10/300 column (GE Healthcare), equilibrated with buffer E (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM MgCl₂, 10 μM ZnCl₂, and 1 mM DTT). The purified complex solution (0.5 mg ml⁻¹ final concentration) was applied to freshly glow-discharged Au 300 mesh R1.2/1.3 grids (Quantifoil) after adding 3 μl of amylamine, using a Vitrobot Mark IV (FEI) at 4 °C, with a waiting time of 10 s and a blotting time of 4 s under 100% humidity conditions. The grids were then plunge-frozen in liquid ethane cooled to the temperature of liquid nitrogen.

Cryo-EM data were collected using a Titan Krios G3i microscope (Thermo Fisher Scientific), running at 300 kV and equipped with a Gatan Quantum-LS Energy Filter (GIF) and a Gatan K3 Summit direct electron detector in the electron counting mode (The University of Tokyo, Japan). Movies were recorded at a nominal magnification of 105,000×, corresponding to a calibrated pixel size of 0.83Å, with a total dose of approximately 50 electrons per Ų per 48 frames. The data were automatically acquired using the EPU software (Thermo Fisher Scientific), with a defocus range of −0.8 to −1.6 μm, and 3,570 movies were obtained.

Image processing

The data processing was performed with the cryoSPARC v3.3.2 software platform²⁸. The dose-fractionated movies were aligned using patch motion correction, and the contrast transfer function (CTF) parameters were estimated using the Patch-based CTF estimation. From the 3,570 motion-corrected and dose-weighted micrographs, 2,136,853 particles were automatically picked using blob picker in cryoSPARC. The particles were subjected to several rounds of reference-free 2D classifications to create particle sets. The particles were curated by cryoSPARC heterogenous refinement (n = 4), using the map derived from the cryoSPARC ab initio reconstruction as a template. The selected particles were subjected to 3D variability analysis, and the resulting maps with different conformations were used for subsequent heterogeneous refinement. The best class containing 98,483 particles was refined using non-uniform refinement²⁹ after CTF refinement, yielding a map at 3.21 Å resolution, according to the Fourier shell correlation (FSC) = 0.143 criterion³⁰. The local resolution was estimated by cryoSPARC.

Model building and validation

The model was built using the predicted model of the ISDra2 TnpB protein created by AlphaFold2 as the reference³¹, followed by manual model building with COOT³². The model was refined using phenix.real_space_refine ver. 1.20.1³³, with secondary structure and metal coordination restraints. The metal coordination restraints were generated using ReadySet, as implemented in PHENIX. The structure validation was performed using MolProbity in the PHENIX package³⁴. The EMRinger score³⁵ and 3DFSC sphericity³⁶ were calculated by PHENIX and the 3DFSC processing Server (https://3dfsc.salk.edu/upload/info/), respectively. The statistics of the 3D reconstruction and model refinement are summarized in Extended Data Table 1. The cryo-EM density map figures were generated using UCSF ChimeraX³⁷. Molecular graphics figures were prepared using CueMol (http://www.cuemol.org).

Northern blotting analysis

Total RNA was extracted from cells expressing ωRNA or ωRNA-MBP or ωRNA-TnpB with TRIzol LS (Thermo Fisher Scientific) according to the manufacturer’s instructions. TnpB-interacting ωRNA was extracted from the purified TnpB–ωRNA complex with TRIzol LS (Thermo Fisher Scientific) according to the manufacturer’s instructions. Three micrograms of total RNA, 140 ng of in vitro-transcribed ωRNA and 140 ng of TnpB-interacting ωRNA were resolved by electrophoresis on a 10% polyacrylamide gel containing 7 M urea, followed by staining with GelGreen (Biotium). Fluorescence was visualized by an FLA-7000 imaging analyser (Fujifilm). The RNAs were transferred to a Hybond N⁺ membrane (Cytiva) by electroblotting for 1 h at 1.5 mA cm⁻² in 1× TBE using a Transblot Turbo (Bio-Rad), and crosslinked by two rounds of UV irradiation (254 nm, 120 mJ cm⁻²; CL-1000, UVP). The membrane was treated with hybridization buffer (5% PEG 6000 (w/v), 7.5% SDS (w/v), 0.5% casein (w/v), 1 mM EDTA (pH 8.0), and 282 mM sodium phosphate buffer (pH 7.4)) at 52 °C for 1 h, and then subjected to hybridization with 2 pmol of the 5′-³²P-labelled DNA probes at 50 °C overnight. The sequences of DNA probes are as follows: 5′-TTCTTCACTTCGGGATTCTTGAATC-3′ (probe I), 5′-CGTCTCGGTCATGGGTTTCCCCACA-3′ (probe II), 5′-GTCTGAGATTCCCGCAGCCACCAAC-3′ (probe III), 5′-GCAGACCATTGCCCGCCGAAGCAGG-3′ (probe IV), 5′-GGGCGCCAAGGGACTCTTGAACCTC-3′ (probe V) (Supplementary Table 2). The membrane was washed four times with 2× SSC (0.3 M NaCl, 30 mM sodium citrate (pH 7.0)), dried, and exposed to an imaging plate. Radioactivity was visualized by using an FLA-7000 imaging analyser. Uncropped images are available in the Source Data file.

LC–MS analysis

Each band on the gel was cut into cubes smaller than 1 mm³, soaked in 150 μl elution buffer (3 M sodium acetate (pH 5.3), 1 mM EDTA (pH 8.0), and 0.1% SDS), and shaken for 2 h at 37 °C. The buffer was transferred to a new tube. The gel fragments were then shaken with another 150 μl of elution buffer at 37 °C overnight. The elution buffers were combined, and after glycogen addition, the RNA was recovered by ethanol precipitation. The RNA precipitate was dissolved in water and digested by RNase A (Thermo Fisher Scientific), and then analysed by LC–MS. RNA fragment analysis was performed with an UltiMate 3000 RSLCnano system coupled with an Orbitrap Eclipse Tribrid (Thermo Fisher Scientific). One picomole of the RNA digest was diluted with 10 mM triethylammonium acetate, and loaded on a trap column (Acclaim PepMap 100 C18, 100 μm ID × 20 mm, Thermo Fisher Scientific). RNA fragments were separated on an ODS column (HiQ sil C18W-3, 100 μm ID × 100 mm, Techno Alpha) at a 300 nl min⁻¹ flow rate. Separation was started with 99% mobile phase A (0.4 M hexafluoroisopropanol in water) and 1% B (0.4 M hexafluoroisopropanol in 50% methanol) for 10 min, and then B % was increased to 60% by linear gradient over 32 min. Eluents were injected into the ESI source through a nanoESI emitter (LOTUS emitters, FOSSILIONTECH), and ions were scanned by MS in the negative polarity mode.

In vitro DNA cleavage assay

For the in vitro cleavage assay, the TnpB–ωRNA complexes (wild-type or mutants) were purified in a similar manner to that for the complex prepared for the cryo-EM analysis. Protein concentrations were measured using a Bradford Protein Assay Kit (TAKARA). The DNA cleavage activity of TnpB was measured by in vitro DNA cleavage assays. The TnpB–ωRNA complex (2 μl, final concentration 250 nM) was mixed with the 3-kb linearized plasmid target containing the 16-nt target sequence and the TTGAT TAM (8 μl, 100 ng) (Supplementary Table 1), and incubated at 37 or 50 °C for 30 min in 10 μl reaction buffer (20 mM HEPES, pH 7.5, 50 mM KCl, 2 mM MgCl₂, 1 mM DTT, and 5% glycerol). The reaction was stopped by the addition of quench buffer, containing EDTA (20 mM final concentration) and Proteinase K (40 ng). The reaction products were resolved, visualized, and quantified with a MultiNA microchip electrophoresis device (Shimadzu). In vitro cleavage experiments were performed at least three times.

Mammalian genome editing assays

Mammalian cell culture experiments were performed in the HEK293FT cell line, grown in Dulbecco’s modified Eagle’s medium with high glucose, sodium pyruvate, and GlutaMAX (Thermo Fisher), supplemented with 1× penicillin–streptomycin (Thermo Fisher) and 10% fetal bovine serum (VWR Seradigm). All cells were maintained at confluency below 80%. All transfections were performed with Lipofectamine 3000 (Thermo Fisher) in 96-well plates, unless otherwise noted. Cells were plated at approximately 20,000 cells per well 16–20 h prior to transfection, to ensure 90% confluency at the time of transfection. To evaluate indel efficiencies, transfection plasmids were combined with OptiMEM I Reduced Serum Medium (Thermo Fisher) to a total volume of 20 µl per well. Separately, 18.8 µl of OptiMEM was combined with 1.2 µl of Lipofectamine 3000. The plasmid and Lipofectamine solutions were then combined, and 10 µl was pipetted onto each well. Genomic DNA was collected 96 h after transfection by removing the supernatant and resuspending each well in 50 μl of QuickExtract DNA Extraction Solution (Lucigen). Cells were lysed by cycling at 65 °C for 15 min, 68 °C for 15 min, and 95 °C for 10 min. A 3 μl portion of lysed cells was used as the input in each PCR reaction for deep sequencing, and indel frequencies were quantified by CRISPResso2³⁸. Genome-wide off-target analysis was performed using tagmentation-based tag integration site sequencing (TTISS) as described previously²⁵, and sites identified by TTISS were subjected to quantification of indel frequencies in a separate experiment (Supplementary Tables 1 and 2).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.