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Regulation of gene editing using T-DNA concatenation

Nature Plants volume 9, pages 1398–1408 (2023)Cite this article

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Abstract

Transformation via Agrobacterium tumefaciens is the predominant method used to introduce exogenous DNA into plant genomes1,2. Transfer DNA (T-DNA) originating from Agrobacterium can be integrated as a single copy or in complex concatenated forms3,4, but the mechanisms affecting final T-DNA structure remain unknown. Here we demonstrate that inclusion of retrotransposon (RT)-derived sequences in T-DNA can increase T-DNA copy number by more than 50-fold in Arabidopsis thaliana. These additional T-DNA copies are organized into large concatemers, an effect primarily induced by the long terminal repeats (LTRs) of RTs that can be replicated using non-LTR DNA repeats. We found that T-DNA concatenation is dependent on the activity of the DNA repair proteins MRE11, RAD17 and ATR. Finally, we show that T-DNA concatenation can be used to increase the frequency of targeted mutagenesis and gene targeting. Overall, this work uncovers molecular determinants that modulate T-DNA copy number in Arabidopsis and demonstrates the utility of inducing T-DNA concatenation for plant gene editing.

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Fig. 1: T-DNAs featuring RT sequences are concatenated at higher levels in Arabidopsis.
Fig. 2: The repetitive nature of LTRs promotes T-DNA concatenation.
Fig. 3: T-DNA concatenation is dependent on DNA repair.
Fig. 4: T-DNA concatenation increases gene editing efficiency.

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Data availability

The data generated from this study are included within the main figures, extended data and Supplementary Information. Raw sequencing data are deposited at the NCBI SRA (BioProject PRJNA892619). Analysis of the data made use of the TAIR10 Arabidopsis genome (https://www.arabidopsis.org/download/index-auto.jsp%3Fdir%3D%252Fdownload _files%252FGenes%252FTAIR10_genome_release) and the ‘Orthologous gene family’ list sourced from the Dicots PLAZA 5.0 database (https://ftp.psb.ugent.be/pub/plaza/plaza_public_dicots_05/GeneFamilies/genefamily_data.ORTHOFAM.csv.gz). There are no restrictions on data availability. Source data are provided with this paper.

References

  1. Gelvin, S. B. Integration of Agrobacterium T-DNA into the plant genome. Annu. Rev. Genet. 51, 195–217 (2017).

    Article  CAS  PubMed  Google Scholar 

  2. Gelvin, S. B. Plant DNA repair and Agrobacterium T-DNA integration. Int. J. Mol. Sci. https://doi.org/10.3390/ijms22168458 (2021).

  3. Jupe, F. et al. The complex architecture and epigenomic impact of plant T-DNA insertions. PLoS Genet. 15, e1007819 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  4. Pucker, B., Kleinbolting, N. & Weisshaar, B. Large scale genomic rearrangements in selected Arabidopsis thaliana T-DNA lines are caused by T-DNA insertion mutagenesis. BMC Genomics 22, 599 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Chilton, M. D. et al. Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell 11, 263–271 (1977).

    Article  CAS  PubMed  Google Scholar 

  6. Galbiati, M., Moreno, M. A., Nadzan, G., Zourelidou, M. & Dellaporta, S. L. Large-scale T-DNA mutagenesis in Arabidopsis for functional genomic analysis. Funct. Integr. Genomics 1, 25–34 (2000).

    CAS  PubMed  Google Scholar 

  7. Zambryski, P. et al. Tumor DNA structure in plant cells transformed by A. tumefaciens. Science 209, 1385–1391 (1980).

    Article  CAS  PubMed  Google Scholar 

  8. Baltes, N. J., Gil-Humanes, J., Cermak, T., Atkins, P. A. & Voytas, D. F. DNA replicons for plant genome engineering. Plant Cell 26, 151–163 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Kralemann, L. E. M. et al. Distinct mechanisms for genomic attachment of the 5′ and 3′ ends of Agrobacterium T-DNA in plants. Nat. Plants 8, 526–534 (2022).

    Article  CAS  PubMed  Google Scholar 

  10. van Kregten, M. et al. T-DNA integration in plants results from polymerase-θ-mediated DNA repair. Nat. Plants 2, 16164 (2016).

    Article  CAS  PubMed  Google Scholar 

  11. Jacob, Y. et al. Regulation of heterochromatic DNA replication by histone H3 lysine 27 methyltransferases. Nature 466, 987–991 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Davarinejad, H. et al. The histone H3.1 variant regulates TONSOKU-mediated DNA repair during replication. Science 375, 1281–1286 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zaratiegui, M. Cross-regulation between transposable elements and host DNA replication. Viruses https://doi.org/10.3390/v9030057 (2017).

  14. Cavrak, V. V. et al. How a retrotransposon exploits the plant’s heat stress response for its activation. PLoS Genet. 10, e1004115 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Ito, H. et al. An siRNA pathway prevents transgenerational retrotransposition in plants subjected to stress. Nature 472, 115–119 (2011).

    Article  CAS  PubMed  Google Scholar 

  16. Wolter, F., Klemm, J. & Puchta, H. Efficient in planta gene targeting in Arabidopsis using egg cell-specific expression of the Cas9 nuclease of Staphylococcus aureus. Plant J. 94, 735–746 (2018).

    Article  CAS  PubMed  Google Scholar 

  17. Bechtold, N., Ellis, J. & Pelletier, G. In planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C. R. Acad. Sci. III 316, 1194–1199 (1993).

    CAS  Google Scholar 

  18. Alonso, J. M. et al. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653–657 (2003).

    Article  PubMed  Google Scholar 

  19. McElver, J. et al. Insertional mutagenesis of genes required for seed development in Arabidopsis thaliana. Genetics 159, 1751–1763 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Sessions, A. et al. A high-throughput Arabidopsis reverse genetics system. Plant Cell 14, 2985–2994 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kamp, J. A., van Schendel, R., Dilweg, I. W. & Tijsterman, M. BRCA1-associated structural variations are a consequence of polymerase theta-mediated end-joining. Nat. Commun. 11, 3615 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Jacob, Y. et al. Selective methylation of histone H3 variant H3.1 regulates heterochromatin replication. Science 343, 1249–1253 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Jacob, Y. et al. ATXR5 and ATXR6 are H3K27 monomethyltransferases required for chromatin structure and gene silencing. Nat. Struct. Mol. Biol. 16, 763–768 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Nishizawa-Yokoi, A. et al. Agrobacterium T-DNA integration in somatic cells does not require the activity of DNA polymerase θ. New Phytol. 229, 2859–2872 (2021).

    Article  CAS  PubMed  Google Scholar 

  25. Inagaki, S. et al. Arabidopsis TEBICHI, with helicase and DNA polymerase domains, is required for regulated cell division and differentiation in meristems. Plant Cell 18, 879–892 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Hussmann, J. A. et al. Mapping the genetic landscape of DNA double-strand break repair. Cell 184, 5653–5669.e25 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Budzowska, M. et al. Mutation of the mouse Rad17 gene leads to embryonic lethality and reveals a role in DNA damage-dependent recombination. EMBO J. 23, 3548–3558 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Wang, Q. et al. Rad17 recruits the MRE11-RAD50-NBS1 complex to regulate the cellular response to DNA double-strand breaks. EMBO J. 33, 862–877 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Williams, G. J., Lees-Miller, S. P. & Tainer, J. A. Mre11-Rad50-Nbs1 conformations and the control of sensing, signaling, and effector responses at DNA double-strand breaks. DNA Repair 9, 1299–1306 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Fauser, F. et al. In planta gene targeting. Proc. Natl Acad. Sci. USA 109, 7535–7540 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Scheiffele, P., Pansegrau, W. & Lanka, E. Initiation of Agrobacterium tumefaciens T-DNA processing. Purified proteins VirD1 and VirD2 catalyze site- and strand-specific cleavage of superhelical T-border DNA in vitro. J. Biol. Chem. 270, 1269–1276 (1995).

    Article  CAS  PubMed  Google Scholar 

  32. Ward, E. R. & Barnes, W. M. VirD2 protein of Agrobacterium tumefaciens very tightly linked to the 5′ end of T-strand DNA. Science 242, 927–930 (1988).

  33. Neale, M. J., Pan, J. & Keeney, S. Endonucleolytic processing of covalent protein-linked DNA double-strand breaks. Nature 436, 1053–1057 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Brzezinka, K., Altmann, S. & Baurle, I. BRUSHY1/TONSOKU/MGOUN3 is required for heat stress memory. Plant Cell Environ. 42, 771–781 (2019).

    Article  CAS  PubMed  Google Scholar 

  35. Li, W. et al. The Arabidopsis AtRAD51 gene is dispensable for vegetative development but required for meiosis. Proc. Natl Acad. Sci. USA 101, 10596–10601 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Valuchova, S. et al. Protection of Arabidopsis blunt-ended telomeres is mediated by a physical association with the Ku heterodimer. Plant Cell 29, 1533–1545 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Heacock, M. L., Idol, R. A., Friesner, J. D., Britt, A. B. & Shippen, D. E. Telomere dynamics and fusion of critically shortened telomeres in plants lacking DNA ligase IV. Nucleic Acids Res. 35, 6490–6500 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Heitzeberg, F. et al. The Rad17 homologue of Arabidopsis is involved in the regulation of DNA damage repair and homologous recombination. Plant J. 38, 954–968 (2004).

    Article  CAS  PubMed  Google Scholar 

  39. Samanic, I., Simunic, J., Riha, K. & Puizina, J. Evidence for distinct functions of MRE11 in Arabidopsis meiosis. PLoS ONE 8, e78760 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Feng, W. et al. Large-scale heterochromatin remodeling linked to overreplication-associated DNA damage. Proc. Natl Acad. Sci. USA 114, 406–411 (2017).

    Article  CAS  PubMed  Google Scholar 

  41. Culligan, K., Tissier, A. & Britt, A. ATR regulates a G2-phase cell-cycle checkpoint in Arabidopsis thaliana. Plant Cell 16, 1091–1104 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Curtis, M. D. & Grossniklaus, U. A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol. 133, 462–469 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).

    Article  CAS  PubMed  Google Scholar 

  44. Puizina, J., Siroky, J., Mokros, P., Schweizer, D. & Riha, K. Mre11 deficiency in Arabidopsis is associated with chromosomal instability in somatic cells and Spo11-dependent genome fragmentation during meiosis. Plant Cell 16, 1968–1978 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Molina-Risco, M. et al. Optimizing Agrobacterium-mediated transformation and CRISPR-Cas9 gene editing in the tropical japonica rice variety presidio. Int. J. Mol. Sci. https://doi.org/10.3390/ijms222010909 (2021).

  46. Clemente, T. Nicotiana (Nicotiana tobaccum, Nicotiana benthamiana). Methods Mol. Biol. 343, 143–154 (2006).

    PubMed  Google Scholar 

  47. Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(–Delta Delta C(T)) method. Methods 25, 402–408 (2001).

    Article  CAS  PubMed  Google Scholar 

  48. Wang, S. et al. Spatial organization of chromatin domains and compartments in single chromosomes. Science 353, 598–602 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Rouillard, J. M., Zuker, M. & Gulari, E. OligoArray 2.0: design of oligonucleotide probes for DNA microarrays using a thermodynamic approach. Nucleic Acids Res. 31, 3057–3062 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Sage, D. et al. DeconvolutionLab2: an open-source software for deconvolution microscopy. Methods 115, 28–41 (2017).

    Article  CAS  PubMed  Google Scholar 

  51. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  PubMed  Google Scholar 

  52. Jovtchev, G., Schubert, V., Meister, A., Barow, M. & Schubert, I. Nuclear DNA content and nuclear and cell volume are positively correlated in angiosperms. Cytogenet. Genome Res. 114, 77–82 (2006).

    Article  CAS  PubMed  Google Scholar 

  53. Ollion, J., Cochennec, J., Loll, F., Escude, C. & Boudier, T. TANGO: a generic tool for high-throughput 3D image analysis for studying nuclear organization. Bioinformatics 29, 1840–1841 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Chen, S., Zhou, Y., Chen, Y. & Gu, J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34, i884–i890 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  55. Cock, P. J. et al. Biopython: freely available Python tools for computational molecular biology and bioinformatics. Bioinformatics 25, 1422–1423 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).

    Article  CAS  PubMed  Google Scholar 

  57. Lamesch, P. et al. The Arabidopsis Information Resource (TAIR): improved gene annotation and new tools. Nucleic Acids Res. 40, D1202–D1210 (2012).

    Article  CAS  PubMed  Google Scholar 

  58. Li, H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. Preprint at https://arxiv.org/abs/1303.3997v2 (2013).

  59. Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Van Bel, M. et al. PLAZA 5.0: extending the scope and power of comparative and functional genomics in plants. Nucleic Acids Res. 50, D1468–D1474 (2022).

    Article  PubMed  Google Scholar 

  61. Danecek, P. et al. Twelve years of SAMtools and BCFtools. GigaScience https://doi.org/10.1093/gigascience/giab008 (2021).

  62. Ramirez, F. et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 44, W160–W165 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Jasper, F., Koncz, C., Schell, J. & Steinbiss, H. H. Agrobacterium T-strand production in vitro: sequence-specific cleavage and 5′ protection of single-stranded DNA templates by purified VirD2 protein. Proc. Natl Acad. Sci. USA 91, 694–698 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Kleinboelting, N. et al. The structural features of thousands of T-DNA insertion sites are consistent with a double-strand break repair-based insertion mechanism. Mol. Plant 8, 1651–1664 (2015).

    Article  CAS  PubMed  Google Scholar 

  65. Feng, W. et al. Marker-free quantification of repair pathway utilization at Cas9-induced double-strand breaks. Nucleic Acids Res. 49, 5095–5105 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank all current and former members of the Jacob lab, especially F. Langhammer and G. Villarino, for reagents, discussions and advice; C. Bolick and his staff at Yale for help with plant growth and maintenance; H. Puchta from the Karlsruhe Institute of Technology for his generous gifts of the gene targeting binary plasmids used in this work; and Marco Molina and Mayra Molina (Multi-Crop Transformation Facility, Texas A&M University) for generating the transgenic tobacco plants used in this study. This project was supported by grant R35GM128661 from the National Institutes of Health (Y.J.) and an NIH Director’s New Innovator Award (DP2GM137414 to S.W.).

Author information

Author notes

  1. These authors contributed equally: Lauren Dickinson, Wenxin Yuan.

Authors and Affiliations

  1. Department of Molecular, Cellular and Developmental Biology, Faculty of Arts and Sciences, Yale University, New Haven, CT, USA

    Lauren Dickinson, Wenxin Yuan, Chantal LeBlanc, Geoffrey Thomson & Yannick Jacob

  2. Department of Genetics, Yale School of Medicine, Yale University, New Haven, CT, USA

    Siyuan Wang

  3. Department of Cell Biology, Yale School of Medicine, Yale University, New Haven, CT, USA

    Siyuan Wang

  4. Yale Cancer Center, Yale School of Medicine, Yale University, New Haven, CT, USA

    Yannick Jacob

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Contributions

Y.J. supervised the study and designed the experiments with L.D. and W.Y. All experiments were performed by L.D. and W.Y., except the targeted mutagenesis work (done by C.L.) and the bioinformatic analyses (done by G.T.). S.W. designed the probes for the FISH experiments. Y.J. and C.L. wrote the paper, with contributions from L.D. and W.Y.

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Correspondence to Yannick Jacob.

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Competing interests

A patent application (patent application no. 63/481,276, US Patent and Trademark Office, 24 January 2023), with Y.J., L.D. and W.Y. as inventors, related to the use of repetitive sequences and genetic mutations to regulate T-DNA copy number and improve gene editing, has been filed. The other authors declare no competing interests.

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Nature Plants thanks Avraham Levy, Damon Lisch and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 DNA FISH analysis.

a, DNA-qPCR of ALS in Col-0 (untransformed) and T1 plants used for the FISH experiment (panel b and Fig. 1e). Dots represent DNA samples from individual leaves from the same T1 plant (n = 4 for Col-0, ONSEN RT #1 and ONSEN RT #2, n = 3 for No RT). Horizontal bars indicate the mean. SD is shown. b, FISH in leaf nuclei targeting the T-DNA sequence. Nuclei were stained with DAPI and probes for ALS (green) and NPTII (red). The FISH experiment was performed >3 times with similar results.

Extended Data Fig. 2 Whole genome sequencing analysis of T-DNA insertions.

a, DNA-qPCR of ALS in the No RT and ONSEN RT T1 plants used for whole genome sequencing (panel b, Fig. 1f,g and Fig. 3b). b, Locus diagrams for the identified T-DNA insertions. The coordinates for each insertion are based on the TAIR10 annotation and correspond to the Arabidopsis genomic borders surrounding each identified T-DNA. For each insertion, top lines represent unaltered genomic sequence with annotated genes (shades of navy blue). Red arrows represent insertion points. The bottom lines show the borders of the insertion in more detail, with the identified insertion of binary vector (non-T-DNA region) sequence or T-DNA components shown. Dashed lines represent contiguous T-DNA-associated cassettes. Red bars indicate binary vector sequence (non-T-DNA). Dark blue bars (LB) and light gray bars (pea3A(T)) indicate the 5′ end of the T-DNA construct (though it may be in 5′ or 3′ orientation in the plant genome). Darker gray bars adjacent to the red bars are filler sequences, and the teal bar represents NPTII sequence.

Extended Data Fig. 3 Repetitive sgRNA genes do not contribute to T-DNA concatenation.

a, Schematic representation of the sgRNA gene deletion from the No RT vector. b, DNA-qPCR of ALS in Col-0 (untransformed) and in T1 plants transformed with the No RT plasmid and the No RT plasmid with a sgRNA gene deleted. Each dot represents an individual plant (n = 21 for Col-0, n = 26 individual T1 plants for No RT and No RT, sgRNA gene deletions). Horizontal bars indicate the median. PCol-0 = 0.00000002, ns = not significantly different (Kruskal-Wallis ANOVA followed by Dunn’s test). c. Schematic representation of the sgRNA gene deletion from the ONSEN RT vector. d. DNA-qPCR of ALS in Col-0 (untransformed) and in T1 plants transformed with the ONSEN RT plasmid and the ONSEN RT plasmid with both sgRNA genes deleted. Each dot represents an individual plant (n = 22). Horizontal bars indicate the median. PCol-0 = 0.00000004, ns = not significantly different (Kruskal-Wallis ANOVA followed by Dunn’s test). e. DNA-qPCR of ALS in tobacco plants transformed by biolistic bombardment with particles coated with No RT or ONSEN RT plasmid DNA. Each dot represents an individual plant. Horizontal bars indicate the median. ns = not significantly different (two-tailed Mann-Whitney U test). ns, P > 0.05; ****, P < 0.0001.

Source data

Extended Data Fig. 4 T-DNA junctions support the involvement of TMEJ in T-DNA concatenation.

a, Multiple sequence alignments of junctions between two T-DNA sequences grouped by orientation of the two sequences (RB-LB, LB-LB or RB-RB). Reference sequences were constructed assuming T-DNA molecules began and ended immediately upstream of the endonuclease (VirD2) recognition sequence (5′-CAGGATATATT-3′)63,64. Filler sequences are in red and sequences consistent with microhomology-associated deletions are underlined. If filler sequences have a similar sequence nearby, they (and the nearby sequence) are also underlined. Asterisks indicate identical junctions occurring in independent plants. b, Depiction of T-DNA junctions with another T-DNA or binary vector sequence not immediately internally adjacent to the LB or RB. c. Classification of RB-LB, LB-LB and RB-RB sequences for each T1 line following a procedure previously described65. NHEJ (<4 bp deletions and <5 bp insertions), insertions (≥5 bp with any deletion), non-microhomology (Non-MH; ≥4 bp deletion or <5 bp insertions with microhomologies <2), and microhomology (MH; ≥4 bp deletion with microhomologies ≥2). The latter three are associated with DNA polymerase theta.

Extended Data Fig. 5 T-DNA concatenation is not DNA replication-dependent.

a, DNA-qPCR of ALS in Col-0, atxr5/6, and tsk plants transformed with the ONSEN RT construct. Each dot represents an individual T1 plant (n = 24). Horizontal bars indicate the median. ns = not significantly different (Kruskal-Wallis ANOVA followed by Dunn’s test). b, DNA-qPCR of ALS in DNA extracted from leaves of T1 plants at 16 days and 30 days after germination.

Source data

Extended Data Fig. 6 T-DNA concatenation increases the efficiency of targeted mutagenesis and gene targeting.

a. Gene structure of CRY2. Gray bars represent exons, and blue bars represent regions targeted by sgRNAs. b, c. INDEL frequency of CRY2 PCR products amplified from DNA extracted from leaves of individual T1 plants transformed with either No RT or ONSEN RT constructs. Constructs carried either (b) sgRNA #2 or (c) sgRNA #3. d. DNA-qPCR of ALS in T1 plants with no detectable CRY2 indels (No indels) or detectable indels (Indels) using CRY2 sgRNA #3. Each dot represents an individual T1 plant (n = 20). Horizontal bars indicate the median. *P = 0.0350 (two-tailed Mann-Whitney U test) e. DNA-qPCR of ALS in No RT and ONSEN RT T1 plants (gray) in relation to the percentage of true (non-ectopic) gene targeting rates in the T2 generation (red).

Source data

Extended Data Fig. 7 Model of T-DNA concatenation.

The model builds on the T-DNA integration model from Kralemann et al., 20226. Chromosomal capture of a T-DNA strand 3′ end is mediated by TMEJ. After conversion to a double-stranded T-DNA intermediate, capture of the T-DNA 5′ end is accomplished by removal of the Agrobacterium protein VirD2 by TDP2 or MRE11. TDP2-mediated removal of VirD2 creates blunt-ended DNA at the T-DNA 5′ end that is ligated to the chromosome by NHEJ. In contrast, MRE11, acting as part of the MRN complex (MRE11-RAD50-NBS1; loaded on DNA by RAD1725), removes VirD2 by cutting the T-DNA internally, generating a staggered end at the T-DNA 5′ end. TDP2/NHEJ activity leads to a single T-DNA copy integration (outcome A), while MRE11/TMEJ activity leads to multiple outcomes (B-E), with the simplest one being chromosomal capture of the T-DNA 5′ end (outcome B). Alternatively, the staggered T-DNA 5′ end can facilitate recruitment of additional T-DNA strands for ligation, leading to concatenation. Capture of the 5′ end of an additional T-DNA strand by TDP2/NHEJ instead of MRE11/TMEJ is more likely to terminate the concatenation cycle. In this model, T-DNA features like DNA repeats may increase concatenation levels by increasing the number of available T-DNA strands for integration, and/or their accessibility. ssDNA: single-stranded DNA. dsDNA: double-stranded DNA. Created with BioRender.com.

Supplementary information

Reporting Summary

Supplementary Table 1

Supplementary Table 1. List of probes used for FISH analysis and primers.

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Source Data Extended Data Fig. 6

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Dickinson, L., Yuan, W., LeBlanc, C. et al. Regulation of gene editing using T-DNA concatenation. Nat. Plants 9, 1398–1408 (2023). https://doi.org/10.1038/s41477-023-01495-w

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