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Transposon-encoded nucleases use guide RNAs to promote their selfish spread

Nature volume 622, pages 863–871 (2023)Cite this article

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Abstract

Insertion sequences are compact and pervasive transposable elements found in bacteria, which encode only the genes necessary for their mobilization and maintenance1. IS200- and IS605-family transposons undergo ‘peel-and-paste’ transposition catalysed by a TnpA transposase2, but they also encode diverse, TnpB- and IscB-family proteins that are evolutionarily related to the CRISPR-associated effectors Cas12 and Cas9, respectively3,4. Recent studies have demonstrated that TnpB and IscB function as RNA-guided DNA endonucleases5,6, but the broader biological role of this activity has remained enigmatic. Here we show that TnpB and IscB are essential to prevent permanent transposon loss as a consequence of the TnpA transposition mechanism. We selected a family of related insertion sequences from Geobacillus stearothermophilus that encode several TnpB and IscB orthologues, and showed that a single TnpA transposase was broadly active for transposon mobilization. The donor joints formed upon religation of transposon-flanking sequences were efficiently targeted for cleavage by RNA-guided TnpB and IscB nucleases, and co-expression of TnpB and TnpA led to substantially greater transposon retention relative to conditions in which TnpA was expressed alone. Notably, TnpA and TnpB also stimulated recombination frequencies, surpassing rates observed with TnpB alone. Collectively, this study reveals that RNA-guided DNA cleavage arose as a primal biochemical activity to bias the selfish inheritance and spread of transposable elements, which was later co-opted during the evolution of CRISPR–Cas adaptive immunity for antiviral defence.

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Fig. 1: Pervasive distribution of IS200/IS605-like elements in G. stearothermophilus.
Fig. 2: TnpA catalyses DNA excision for multiple families of IS elements.
Fig. 3: TnpB and IscB target donor joint molecules produced by TnpA.
Fig. 4: Unbiased identification of TnpB/IscB TAM specificity by ChIP–seq and library assays.
Fig. 5: RNA-guided nucleases promote transposon survival via recombination at donor sites.

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Data availability

Next generation sequencing data are available in the National Center for Biotechnology Information (NCBI) Sequence Read Archive: SRX19058888–SRX19058905, SRR23476356–SRR23476358, and SRR24994123 (BioProject Accession: PRJNA925099 and PRJNA986543) and the Gene Expression Omnibus (GSE223127). The published genome used for ChIP–seq analyses was obtained from NCBI (GenBank: CP001509.3). The published genome used for bioinformatics analyses of the G. stearothermophilus genome was obtained from NCBI (GenBank: NZ_CP016552.1). Datasets generated and analysed in the current study are available from the corresponding author upon reasonable request.

Code availability

Custom scripts used for bioinformatics, TAM library analyses and ChIP–seq data analyses are available at GitHub (https://github.com/sternberglab/Meers_et_al_2023).

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Acknowledgements

The authors thank D. R. Gelsinger and A. Bernheim for helpful bioinformatics discussions, L. E. Berchowitz for helpful RNA biology discussions, J. C. Cheong for technical support, L. F. Landweber for qPCR instrument access, and the JP Sulzberger Columbia Genome Center for NGS support. C.M. was supported by NIH Postdoctoral Fellowship F32 GM143924-01A1. M.W.G.W. was supported by a National Science Foundation Graduate Research Fellowship. This research was supported by NSF Faculty Early Career Development Program (CAREER) Award 2239685, and by a generous start-up package from the Columbia University Irving Medical Center Dean’s Office and the Vagelos Precision Medicine Fund (to S.H.S.).

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Author notes

  1. Hoang C. Le

    Present address: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA

  2. Sanjana R. Pesari

    Present address: Biochemistry and Molecular Biophysics Program, University of California, San Diego, CA, USA

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA

    Chance Meers, Sanjana R. Pesari, Florian T. Hoffmann, Jeanine Gezelle, Stephen Tang & Samuel H. Sternberg

  2. Department of Biology, University of Pennsylvania, Philadelphia, PA, USA

    Hoang C. Le

  3. Department of Biological Sciences, Columbia University, New York, NY, USA

    Matt W. G. Walker

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Contributions

C.M. and S.H.S. conceived and designed the project. C.M. performed most experiments, with assistance from S.R.P. on plasmid interference assays, and F.T.H. for transposon excision assays and ChIP–seq experiments and analyses. C.M. and H.C.L. performed all bioinformatics analyses. M.W.G.W. assisted with TAM library design and NGS analysis. J.G. assisted with plasmid interference assays. S.T. performed G. stearothermophilus RNA-seq experiments. C.M. and S.H.S. discussed the data and wrote the manuscript, with input from all authors.

Corresponding author

Correspondence to Samuel H. Sternberg.

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Competing interests

Columbia University has filed US Patent Application Number 63/379,082 related to this work, for which C.M. and S.H.S. are inventors. S.H.S. is a co-founder and scientific advisor to Dahlia Biosciences, a scientific advisor to CrisprBits and Prime Medicine, and an equity holder in Dahlia Biosciences and CrisprBits.

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Extended data figures and tables

Extended Data Fig. 1 Bioinformatic analyses of IscB and TnpB homologs.

a, Phylogenetic tree of IscB and IsrB protein homologs; IscB contains HNH and RuvC nuclease domains, whereas IsrB lacks the HNH nuclease. Genetic neighborhood analyses demonstrate that most homologs are encoded proximal to a predicted ωRNA (inner ring), whereas the vast majority do not reside near a predicted tyrosine-family TnpA transposase gene (outer ring). The GstIscB homolog encoded by ISGst6 used in this study is indicated. Bootstrap values are indicated for major nodes. b, Schematic of a non-autonomous IS element encoding IscB and its associated ωRNA; a structural covariation model is shown in the inset (top). The red rectangle and dotted black line indicate the transposon boundaries, and the guide portion of the ωRNA is shown in blue. LE and RE, transposon left end and right end. c, Orientation bias of the nearest upstream ORFs to the indicated protein-coding gene (iscB, tnpB, or IS630 transposase), demonstrating that IS elements encoding IscB are preferentially integrated (or retained) in an orientation matching that of the upstream gene. The y-axis indicates the frequency of ORFs containing the same orientation, at a distance from the gene start codon defined by the x-axis. 242 bp represents the average length of IscB-associated ωRNAs upstream of IscB ORF. The spike at ~0-bp for TnpB corresponds to IS elements that encode adjacent/overlapping tnpA and tnpB genes. IS630 transposase genes are included as a representative gene from unrelated transposable elements. d, Phylogenetic tree of TnpB homologs, with bootstrap values shown for major nodes. Genetic neighborhood analyses demonstrate that most homologs are encoded proximal to a predicted ωRNA (inner ring), whereas the vast majority do not reside near a predicted tyrosine- or serine-family TnpA transposase genes (outer rings). Interestingly, TnpB homologs are associated with two distinct transposase families in prokaryotes: tyrosine transposases (denoted TnpA (Y)) within IS200/605-family elements, and serine transposases (denote TnpA (S)) within IS607-family elements. GstTnpB homologs used in this study are highlighted, along with the predicted structures of their associated ωRNAs based on covariance modeling. The ωRNA encoded by ISGst2 did not show strong covariation in structure and was therefore omitted. e, Read coverage for RNA-seq data from G. stearothermophilus strain DSM 458, demonstrating expression of putative ωRNAs from each of the indicated ISGst families. ISGst5 is a PATE-like element that lacks any protein-coding ORFs. Other TnpB-associated ωRNAs are encoded within/downstream of the ORF, whereas IscB-associated ωRNAs are encoded upstream of the ORF.

Extended Data Fig. 2 Classification of IS605-family elements encoded by G. stearothermophilus strain DSM 458.

a, DNA multiple sequence alignment of transposon left ends (LE) for IS200/IS605-family elements from G. stearothermophilus. The weblogo (top) is built from 47 unique elements (Supplementary Figs. 2 & 3), and one representative sequence from each family is shown below, with the TAM highlighted in yellow and DNA guide sequences highlighted in red. Nucleotides highlighted in black exhibit covarying mutations, relative to ISGst2. TAM, transposon-adjacent motif; the dotted red line indicates the transposon boundary. b, DNA multiple sequence alignment of transposon right ends (RE) for IS200/IS605-family elements from G. stearothermophilus, shown as in a. TEM, transposon-encoded motif is shown in orange. c, Phylogenetic tree of ISGst elements based on the transposon left end. Each colored clade encodes an associated TnpB/IscB protein homolog and is flanked by the indicated TAM sequence. d, Phylogenetic tree of ISGst elements based on the transposon right end, shown as in b but with TEM sequence in lieu of TAM. e, Schematic of PATEs (palindrome-associated transposable elements) related to ISGst2 and ISGst5, which contain similar transposon ends but no protein-coding genes. The percent sequence identity between shaded regions (black) is shown, as are the genomic accession IDs and coordinates.

Extended Data Fig. 3 Specificity and efficiency of transposon DNA excision by TnpA.

a, Schematic of heterologous E. coli transposon excision assay. Plasmids encode TnpA and a mini-transposon (Mini-Tn) substrate, whose loss is monitored by PCR using the indicated primers. The expected sizes of PCR products generated from donor joints that are produced upon religation of flanking sequences are shown, for both ISGst2 and H. pylori IS608. b, TnpA homologs do not cross-react with distinct IS elements, as assessed by analytical PCR. Cell lysates were tested after overnight expression of TnpA in combination with a mini-Tn substrate from either G. stearothermophilus (G) or H. pylori (H), and PCR products were resolved by agarose gel electrophoresis. M refers to catalytically inactive mutants. Note that HpyTnpA is substantially more active for DNA excision than GstTnpA under the tested conditions. U, unexcised; E, excised. c, Schematic of qPCR assay to quantify excision frequencies, in which one of the two primers anneals directly to the donor joint formed upon mini-Tn excision and religation. d, Comparison of simulated excision frequencies, generated by mixing clonally excised and unexcised lysate in known ratios, versus experimentally determined integration efficiencies measured by qPCR. e, qPCR-based quantification of TnpA-mediated excision of an ISGst2 mini-Tn substrate in E. coli. Excised refers to a cloned excision product; TnpAM denotes a TnpA mutant (Y125A); ND, not detected above a 0.0001% threshold. Bars indicate mean ± standard deviation (n = 3). f, Schematic of mini-Tn for ISGst3 element, highlighting the subterminal palindromic transposon ends located on the top strand (top). Transposon-adjacent and transposon-encoded motifs (TAM and TEM) are shown in yellow and orange, respectively; DNA guides are shown in orange, and their putative base-pairing interactions are indicated; dotted lines indicate transposon boundaries and thus the sites of ssDNA cleavage and religation. Sanger sequencing of excision events confirms the identity of the expected donor joint product formed upon transposon loss (bottom). Sanger sequencing results are duplicated from Fig. 2d. g, Schematic and Sanger sequencing data as in f, but for a modified ISGst3 substrate containing TEM mutations. Experimentally detected products erroneously excise at an alternative, aberrant TEM-like sequence located outside of the native transposon boundary (orange), presumably because of the need to maintain cognate base-pairing between the DNA guide (orange and TEM (blue).

Extended Data Fig. 4 Mating-out assay to monitor transposition of ISGst3.

a, Schematic of mating-out assay, in which transposition events into the F-plasmid are monitored via drug selection. E. coli donor cells carrying an F-plasmid were transformed with a plasmid encoding either TnpA and ISGst3-derived mini-Tn (pDonor1) or an autonomous ISGst3 element with tnpA and tnpB (pDonor2). After induction of TnpA, conjugation was used to transfer the F-plasmid into the recipient strain, and transposition events were quantified by selecting for recipient cells (RifR) containing spectinomycin (F+) and kanamycin (mini-Tn+) resistance. b, Transposition frequency of ISGst3 mini-Tn deriving from pDonor1 into the F-plasmid was measured with and without tnpA. Bars indicate mean ± s.d. (n = 6). c, Drug-selected cells from mating-out assays from pDonor1 contain TAM-proximal IS insertions, as evidenced by long-read Nanopore sequencing. A genetic map of the F-plasmid is shown, along with the location of distinct ISGst3-derived mini-Tn integration events. The insets show a zoom-in view of each integration site at the nucleotide level, with the TAM highlighted in yellow and the integration site denoted by an arrow. d, Transposition frequency of an autonomous ISGst3 deriving from pDonor2 into the F-plasmid was measured with active and catalytically inactive forms of TnpA and TnpB. M, TnpA Y125A mutant; d, TnpB D196A mutant; ND, not detected. Bars indicate mean ± s.d. (n = 3).

Extended Data Fig. 5 Optimization and testing of DNA cleavage parameters with TnpB/IscB nucleases.

a, Promoter screen to optimize conditions for E. coli-based interference assays using plasmid-encoded ωRNA and TnpB2. P1 indicates the promoter for ωRNA expression, whereas P2 indicates the promoter for TnpB expression. Transformants with a targeting (T) or non-targeting (NT) ωRNA-pTarget combination were serially diluted, plated on selective media, and cultured at 37 °C for 24 h. b, Results from plasmid interference assays with HpyTnpB (IS608) and DraTnpB (ISDra2) using ωRNAs that target native donor joint products, which revealed an absence of activity for HpyTnpB. Experiments were performed as in a. c, DNA cleavage by TnpB2 is highly sensitive to TAM mutations, as assessed by plasmid interference assays. Data are shown as in a, with the indicated TAM sequences; TTTAT denotes the WT TAM, and NT denotes a non-targeting control. d, DNA cleavage by IscB is highly sensitive to TAM mutations, as assessed by plasmid interference assays. Data are shown as in a, with the indicated TAM sequences; TTCAT denotes the WT TAM, and NT denotes a non-targeting control. e, TnpB2 is only active for targeted genomic DNA cleavage using select ωRNAs, as assessed by genome targeting assays. Transformants with a non-targeting (NT) or one of three lacZ-targeting guides (T1–T3) were serially diluted, plated on selective media, and cultured at 37 °C for 24 h. f, Schematic of E. coli-based genome targeting assay, in which RNA-guided DNA cleavage of lacZ by TnpB results in cell death. Guides were designed to target three sites within the lacZ coding sequence. g, TnpB1 is active for targeted genomic DNA cleavage at elevated temperatures, as assessed by genome targeting assays. Transformants with a non-targeting (NT) or one of three lacZ-targeting guides (T1–T3) were serially diluted, plated on selective media, and cultured at 37 °C or 45 °C for 24 h, as shown. h, Schematic of E. coli-based plasmid interference assay using a native ISGst3 element. The transposon was cloned from G. stearothermophilus gDNA onto the pEffector plasmid, with 40 bp of flanking sequence to preserve the natural ωRNA. Targeted cleavage of pTarget results in a loss of kanamycin resistance and cell lethality on selective LB-agar plates. i, TnpB2 encoded by a native ISGst3 transposon is active for targeted cleavage of its donor joint. Transformants with a targeting (T) or non-targeting (NT) ωRNA-pTarget combination were serially diluted, plated on selective media, and cultured at 37 °C for 24 h. dTnpB2 contains an inactivating D196A mutation, whereas ΔTnpB2 contains a stop codon at codon 32. j, Schematic of E. coli-based genome targeting assay as in e, but with pEffector containing a native ISGst3 transposon cloned into lacZ. RNA-guided DNA cleavage of genomic lacZ results in cell death. k, TnpB2 encoded by a native ISGst3 transposon is active for targeted genomic cleavage of a donor joint mimic. Transformants were serially diluted, plated on selective media, and cultured at 37 °C for 24 h. dTnpB2 contains an inactivating D196A mutation, whereas ΔTnpB2 contains a stop codon at codon 32.

Extended Data Fig. 6 Off-target ChIP-seq DNA binding analyses.

a, ChIP-seq experiments reveal recruitment of dIscB to the target site (blue triangle) with a targeting ωRNA. Genome-wide representation of ChIP-seq data for dIscB reshown from Fig. 4b, with addition of a second replicate. Representative off-target sites for dIscB identified by MACS3 are highlighted (OT1-4) and analyzed in the right panel. The middle panel highlights analysis of off-target binding events using MEME-ChIP, as shown in Fig. 4b. Motifs shared by off-target peaks reveal conserved TAM sequences and little conservation of the adjacent seed sequence. The sequence of the 5′ end of the corresponding ωRNA is shown below each motif. n indicates the number of peaks contributing to the motif and their percentage of total peaks called by MACS3; E, E-value significance of the motif generated from the MEME-ChIP analysis. DNA sequences corresponding to the on-target and off-target sites are shown on the right, with TAM (yellow) and mismatches (orange) highlighted. b, ChIP-seq experiments reveal recruitment of dTnpB2 to the target site (blue triangle) with a targeting ωRNA. Data shown as in a. Similar to dIscB, dTnpB2 shows limited seed sequence requirements. c, ChIP-seq experiments reveal recruitment of dCas9 to the target site (blue triangle) with a targeting ωRNA. Data shown as in a. Analysis of off-target sites reveal a short (3–4 nt) seed sequence adjacent to the PAM motif. d, ChIP-seq experiments reveal recruitment of dCas12a to the target site (blue triangle) with a targeting ωRNA. Data shown as in a. Analysis of off-target sites reveals a short (5–7 nt) seed sequence adjacent to the PAM motif.

Extended Data Fig. 7 qPCR analysis of IS element loss upon TnpA and TnpB co-expression.

a, TnpB promotes transposon retention at the donor site, as assessed by blue-white colony screening for the experiment in Fig. 5a–c; images of representative LB-agar plates are shown. TnpAM, TnpA Y125A mutant; dTnpB, TnpB D196A mutant. b, Schematic of qPCR-based strategy for quantifying excision. Primers are designed to flank the donor joint generated upon excision and religation. Selective PCR conditions with a shortened extension time allows for reduced amplification of the starting locus containing the mini-Tn. rssA was used as a reference gene for ΔCq calculations. c, Comparison of simulated excision frequencies, generated by mixing clonally excised and unexcised lysates in known ratios, versus experimentally determined integration efficiencies measured by qPCR. d, qPCR-based quantification of transposon excision for experiments presented in Fig. 5a–c. TnpA was present in all conditions marked with orange bars, and Empty refers to an experiment lacking TnpB; Excised and Unexcised Marker labels shown in blue correspond to clonal control strains with either wild-type lacZ or mini-Tn-interrupted lacZ, respectively. The detection limit is based on qPCR experiments tested on simulated excision frequency samples shown in b. Bars indicate mean ± s.d. (n = 3–9). e, Images of representative LB-agar plates accompanying the experiments presented in Fig. 5d–f, highlighting the roles of TnpA and TnpB in transposon maintenance and spread via recombination. TnpAM, TnpA Y125A mutant; dTnpB, TnpB D196A mutant.

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Meers, C., Le, H.C., Pesari, S.R. et al. Transposon-encoded nucleases use guide RNAs to promote their selfish spread. Nature 622, 863–871 (2023). https://doi.org/10.1038/s41586-023-06597-1

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