RNA Sequencing, IS621 Excision Analysis and In Vitro Recombination Methods

RNA Sequencing and Analysis

Escherichia coli BL21(DE3) cells (NEB, C2527H), which lack endogenous IS621 elements, were transformed with plasmids encoding either the RE–LE junction or the LE–recombinase–RE region of the IS621 element. The transformed cells were plated on lysogeny broth (LB) agar containing kanamycin and incubated overnight at 37 °C. Colonies were scraped from the plates, and total RNA was extracted using a Direct-zol RNA Miniprep Kit (Zymo Research).

To evaluate the expression of IS621 elements encoded in the E. coli genome, RNA was extracted from One Shot E. coli Mach1 T1^R cells (F^– φ80lacZΔM15 ΔlacX74 hsdR(rK^–, mK⁺) ΔrecA1398 endA1 tonA; Thermo Fisher Scientific, C862003). Cells were harvested from bacterial plates 24 h after plating and incubation at 37 °C. RNA was isolated using the Direct-zol RNA Miniprep Kit.

RNA sequencing was performed on a NovaSeq X Plus platform in collaboration with Novogene Research Services. Paired-end RNA-seq reads from each sample were aligned to the corresponding plasmid and genomic reference sequences using Burrows–Wheeler Aligner-MEM¹⁷. SAMtools¹⁸ was used to filter aligned reads originating from the strand encoding the bRNAs.

For each locus of interest, the Pysam Python module was used to calculate single-base coverage depth from the filtered reads. A quality threshold of 0 was applied. Because the E. coli Mach1 genome contains three highly homologous IS621 loci, many reads mapping to these regions were expected to lack unique assignment to a single locus. Among reads mapped to any of the three IS621 loci, read pairs in which either mate had a mapping quality of zero were classified as nonspecific. These reads could map equally well to other regions of the reference sequence. Coverage from nonspecific reads was quantified separately from coverage generated by reads that mapped specifically to one of the three IS621 loci.

Detection of Plasmid-Based IS621 Excision by PCR

Plasmids containing either the wild-type bRNA, representing the pre-transposon strand exchange (pre-TSE) state for excision, or the engineered bRNA, representing the post-TSE state, were transformed into E. coli BL21(DE3). Because this strain lacks endogenous IS621 elements, all detected excision events originated from the plasmid-encoded constructs.

For constructs without a T7 promoter upstream of the bRNA, bacteria were plated on LB agar containing kanamycin and incubated overnight at 37 °C. For constructs containing a T7 promoter, the agar was supplemented with 0.07 mM IPTG. Colonies were scraped from the plates, and plasmid DNA was extracted using a QIAprep Spin Miniprep Plus Kit (Qiagen).

Primers were designed to span the post-excision LT–RT junction remaining after IS621 element excision from the plasmid, generating a 110-bp PCR product. The primers used to detect circularized IS621 intermediates from the Mach1 genome were also used to confirm circular intermediate formation from plasmid-borne constructs, producing a 725-bp product.

PCR amplification was performed using the protocol described below for native IS621 excision. PCR products were separated on 2% agarose gels and visualized with SYBR Gold (Thermo Fisher Scientific). All amplified products were verified by Sanger sequencing.

Detection of Circular IS621 Intermediates and Post-Excision Sites

Genomic DNA was isolated from freshly purchased aliquots of E. coli strains grown in liquid culture using Zymo Quick-DNA Miniprep Plus Kits, according to the manufacturer’s instructions. Whole-genome sequencing was performed at 100× coverage using the Plasmidsaurus whole-genome nanopore sequencing service. This analysis confirmed that the One Shot Mach1 E. coli T1^R strain contains three IS621 copies, whereas the BL21(DE3) strain contains no IS621 copies.

Bacteria were plated on LB agar and incubated overnight at 37 °C. Colonies were then scraped from the plates, and total DNA was extracted using a Zymo Quick-DNA Miniprep Plus Kit.

Primers were designed to match all three IS621 recombinase-coding sequences in the Mach1 genome. Therefore, PCR amplification occurred only from excised and circularized IS621 sequences originating from any of the three genomic loci. Additional primers were designed to anneal to the junction remaining after IS621 excision at all three loci. Amplification with a downstream primer generated a locus-specific product.

PCR reactions were performed using Platinum SuperFi 2× Master Mix (Thermo Fisher Scientific) under the following conditions:

• 98 °C for 120 s;
• 25 cycles of 98 °C for 10 s, 65 °C for 15 s and 72 °C for 15 s;
• 72 °C for 2 min.

PCR products were separated on 2% agarose gels and visualized using SYBR Gold.

In Vitro IS621 Recombination Measurement by qPCR

DNA substrates consisting of 51-bp LH, 121-bp RH, 86-bp tDNA and 86-bp dDNA sequences were purchased from IDT as single-stranded DNA. The substrates were annealed by heating to 95 °C and then slowly cooling to 4 °C over 1 h in a thermocycler.

DNA substrates at concentrations of 0.25–0.5 µM were combined with the IS621–bRNA complex at 10 µM in a 20-µl reaction containing 20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 5 mM MgCl₂ and 1 mM dithiothreitol (DTT). Reactions were incubated at 37 °C for 2 h.

Reactions were quenched with 50 mM EDTA, treated with 8 µg RNase A (NEB) at 50 °C for 1 h and then treated with 3 units of Proteinase K at 37 °C for 1 h. Following RNA and protein digestion, DNA was purified using a DNA Clean & Concentrator-5 kit (Zymo Research) and eluted with nuclease-free water preheated to 75 °C.

For quantitative PCR analysis, reaction dilutions were prepared and analysed on a LightCycler 480 II instrument (Roche). Primers and PrimeTime qPCR probes were purchased from IDT and used at a final concentration of 0.5 µM with TaqMan Fast Advanced qPCR Master Mix.

In vitro recombination products were expected to contain an unligated bottom strand, with the two nucleotides immediately 3′ to the core mismatched with the top strand after the bottom-strand exchange step. To accurately quantify reaction efficiency while accounting for polymerase processivity through lesions and primer binding near nicks, qPCR signals were compared with standard curves generated using known quantities of 121-bp RH (LD–RT insertion product) and 86-bp target (LT–RT excision product) DNA.

Each standard contained an unligated bottom strand with the expected mismatches adjacent to the core. Standards were prepared by annealing the 121- or 86-nucleotide top strand of RH or target DNA with two oligonucleotides. The RH bottom strand consisted of 56- and 65-nucleotide oligonucleotides, whereas the target bottom strand consisted of 56- and 30-nucleotide oligonucleotides.

In Vivo IS621 Excision Efficiency Measurement by qPCR

To measure IS621 excision efficiency in E. coli, plasmids encoding either the wild-type bRNA, representing the pre-TSE state for excision, or the engineered bRNA, representing the post-TSE state, were transformed into chemically competent BL21(DE3) cells. For each transformation, 100 ng of sequence-verified plasmid was used. Nanopore sequencing confirmed that the input plasmid populations lacked pre-excised molecules.

Transformed cells were recovered in super optimal broth (SOC) medium for 1 h at 37 °C, plated on kanamycin-selective agar and incubated overnight at 37 °C. All colonies from a single transformation plate were harvested in bulk by scraping them into liquid medium. This approach provided a population-level estimate of excision efficiency across thousands of independent clonal lineages and minimized bias caused by stochastic variation between individual colonies.

Total plasmid DNA was extracted from each pooled sample using a QIAprep Spin Miniprep Plus Kit (Qiagen). qPCR was performed using 400 ng of the extracted plasmid DNA. Primers and probes were designed to amplify the kanamycin-resistance marker (HEX), which served as a measure of total plasmid abundance, and the post-excision LT–RT junction (FAM), which was detected on plasmids that had undergone IS621 excision.

Multiplexed TaqMan qPCR was calibrated using a control plasmid containing both the post-excision junction and the kanamycin-resistance marker. This control was used to determine the relative amplification efficiencies of the two probes. The resulting inter-channel correction factor was applied when calculating excision efficiency from experimental samples. At least three independent transformations were performed to confirm reproducibility of the population-level measurement.

IS621 Recombinase Protein and bRNA Preparation

The IS621 recombinase protein was prepared as described previously². Briefly, the IS621 gene was cloned into a modified pFastBac1 vector encoding an N-terminal His₆–Twin-Strep tag and an HRV3C cleavage site. Sf9 cells were infected with baculovirus and cultured at 27 °C for 48 h.

Cells were harvested and lysed, and the soluble fraction was purified by Strep-Tactin affinity chromatography followed by size-exclusion chromatography. The purified protein was concentrated and stored at −80 °C in buffer containing 20 mM HEPES-NaOH, pH 7.5, 500 mM NaCl, 2 mM MgCl₂, 1 mM DTT and 10% glycerol.

bRNAs were transcribed in vitro using T7 RNA polymerase and purified by 10% denaturing polyacrylamide gel electrophoresis containing 7 M urea. bRNA sequences are provided in Supplementary Table 1.

In Vitro Recombination Assays with Fluorescent DNA Substrates

DNA substrates consisting of 38-bp LH, 44-bp RH, 38-bp tDNA and 44-bp dDNA sequences were purchased from Eurofins Genomics. LH and tDNA substrates were labelled with Cy5 at the 5′ ends of the top strands.

For excision reactions, LH and RH substrates were used. For insertion reactions, tDNA and dDNA substrates were used. DNA substrates at 0.4 µM were mixed with a pre-incubated IS621–bRNA complex at 2 µM in a 10-µl reaction containing 20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 5 mM MgCl₂ and 1 mM DTT. Reactions were incubated at 37 °C for 1 h.

Reaction mixtures were treated with Proteinase K and incubated at 95 °C for 2 min in denaturing buffer containing 7 M urea. Samples were analysed on an 18% TBE–urea denaturing gel, and fluorescent signals were imaged using a FUSION Solo S system (Vilber Bio Imaging).

Preparation of the IS621 Excision Complex

The IS621 excision complex was reconstituted by mixing purified IS621 recombinase with a 177-nucleotide bRNA and LH and RH DNA substrates containing 4-nucleotide mismatches on the top strands. The bRNA used for in vitro transcription included a 5′ GGG leader sequence.

bRNAs corresponding to the pre-TSE and post-TSE states were used to reconstitute the respective excision complexes. The resulting complexes were purified by size-exclusion chromatography on a Superose 6 Increase 10/300 column (Cytiva) equilibrated with buffer containing 20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 5 mM MgCl₂ and 1 mM DTT.

Purified excision complexes were concentrated to 0.5–1 mg ml⁻¹ using an Amicon Ultra-4 centrifugal filter unit with a 50-kDa molecular-weight cutoff (Millipore). Protein concentrations were determined using the Pierce 660-nm Protein Assay Reagent.

Cryo-EM Data Collection and Image Processing

For cryo-electron microscopy (cryo-EM), Quantifoil Holey Carbon Grids (R1.2/1.3, Au, 300 mesh; SPT Labtech) were glow-discharged in low-pressure air at a current of 10 mA using a PIB-10 system (Vacuum Device).

Excision complex samples were applied to freshly glow-discharged grids using a Vitrobot Mark IV system (Thermo Fisher Scientific) at 4 °C. The waiting time was 10 s, the blotting time was 6 s and the relative humidity was 100%. Grids were plunge-frozen in liquid ethane cooled by liquid nitrogen.

Data were collected using a Titan Krios G3i transmission electron microscope (Thermo Fisher Scientific) operating at 300 kV and equipped with a Gatan Quantum-LS energy filter and a Gatan K3 Summit direct electron detector. Images were acquired at a nominal magnification of 105,000×, corresponding to a calibrated pixel size of 0.83 Å per pixel.

Movies were dose-fractionated into 50 frames at a detector dose rate of 7.8 e⁻ px⁻¹ s⁻¹ in correlated double-sampling mode. The total accumulated exposure was 50 e⁻ Å⁻². Automated data collection was performed using the image-shift method in EPU software (Thermo Fisher Scientific), with a defocus range of −0.8 to −2.0 µm.

Image processing was performed using cryoSPARC v.4.4.0¹⁹. Dose-fractionated movies were aligned using Patch Motion Correction, and contrast transfer function (CTF) parameters were estimated by patch-based CTF estimation.

For the pre-TSE bRNA excision complex, particles were automatically picked using Blob Picker and Template Picker. Reference-free two-dimensional classification was then used to curate particle sets. Additional particle curation was performed through multiple rounds of Heterogeneous Refinement. The best particle class was refined using Homogeneous Refinement, producing a map at 2.71-Å resolution.

Reference-Based Motion Correction followed by Non-Uniform Refinement with CTF optimization produced a map at 2.44-Å resolution according to the Fourier shell correlation (FSC) = 0.143 criterion²⁰ using a tight mask. However, the final reported resolution was 2.53 Å based on the FSC calculated using a softer refinement mask. Local resolution was estimated using BlocRes in cryoSPARC.

For the post-TSE bRNA excision complex, particles were automatically picked using Template Picker, followed by reference-free two-dimensional classification. Additional curation was performed through several rounds of Heterogeneous Refinement. The best particle class was refined using Non-Uniform Refinement, yielding a map at 2.62-Å resolution.

Reference-Based Motion Correction followed by Non-Uniform Refinement with CTF optimization produced a map at 2.41-Å resolution using a tight mask, according to the FSC = 0.143 criterion. The final reported resolution was 2.55 Å based on the FSC calculated with a softer refinement mask.

Model Building and Validation

Models of the IS621 excision complexes were manually built using COOT²¹, with the IS621 insertion complex structure (PDB ID: 8WT6) used as the initial model. Models were refined against unsharpened half-maps using Servalcat²² and validated with MolProbity²³.

Statistics for three-dimensional reconstruction and model refinement are summarized in Extended Data Table 1. Cryo-EM density maps were calculated using UCSF ChimeraX²⁴, and molecular graphics were prepared with CueMol (www.cuemol.org).

Microscale Thermophoresis Binding Analysis

Microscale thermophoresis (MST) experiments were performed using a Monolith NT.115 Pico Series instrument (NanoTemper Technologies) with premium capillaries. IS621 recombinase was labelled using a RED-MALEIMIDE 2nd Generation cysteine-reactive kit (NanoTemper Technologies), according to the manufacturer’s instructions.

The labelled protein was diluted in buffer containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 5 mM MgCl₂, 1 mM DTT and 0.01% Tween 20. Ligands were prepared by dilution in the same buffer.

For ligand preparation, DNA was purchased from IDT and annealed in buffer containing 10 mM Tris, pH 8.0, 5 mM MgCl₂ and 5 mM KCl. To determine the binding affinities of the IS621–RNA complex for tDNA, dDNA, LH and RH substrates, the complex was used at 20 nM and incubated with serial dilutions of each DNA ligand ranging from 0.3 nM to 10 µM.

MST measurements were performed at 37 °C using 8% LED excitation and medium MST power in Pico-RED excitation mode. Data were analysed with the NanoTemper MO.Affinity Analysis software package, and raw measurements were plotted in Prism for visualization.

Reporting Summary

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

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