Regulation of gene editing using T

Mar 30, 2024

Nature Plants (2023)Cite this article

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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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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.

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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.).

These authors contributed equally: Lauren Dickinson, Wenxin Yuan.

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

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

Siyuan Wang

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

Siyuan Wang

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

Yannick Jacob

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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.

Correspondence to Yannick Jacob.

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.

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

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

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.

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

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.

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

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

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

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

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Received: 31 October 2022

Accepted: 18 July 2023

Published: 31 August 2023

DOI: https://doi.org/10.1038/s41477-023-01495-w

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