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Precise deletion, replacement and inversion of large DNA fragments in plants using dual prime editing

Nature Plants volume 11, pages 191–205 (2025) Cite this article

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Abstract

Precise manipulation of genome structural variations holds great potential for plant trait improvement and biological research. Here we present a genome-editing approach, dual prime editing (DualPE), that efficiently facilitates precise deletion, replacement and inversion of large DNA fragments in plants. In our experiments, DualPE enabled the production of specific genomic deletions ranging from ~500 bp to 2 Mb in wheat protoplasts and plants. DualPE was effective in directly replacing wheat genomic fragments of up to 258 kb with desired sequences in the absence of donor DNA. Additionally, DualPE allowed precise DNA inversions of up to 205.4 kb in wheat plants with efficiencies of up to 51.5%. DualPE also successfully edited large DNA fragments in the dicots Nicotiana benthamiana and tomato, with editing efficiencies of up to 72.7%. DualPE thus provides a precise and efficient approach for large DNA sequence and chromosomal engineering, expanding the availability of precision genome-editing tools for crop improvement.

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Fig. 1: Overview of DualPE-mediated large DNA deletions, replacements and inversions.
The alternative text for this image may have been generated using AI.
Fig. 2: Precise large DNA deletions in wheat using the DualPE system.
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Fig. 3: DualPE-mediated sequence replacement in wheat.
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Fig. 4: Use of DualPE to generate large DNA inversions in wheat.
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Fig. 5: DualPE enables large DNA editing in N. benthamiana.
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Fig. 6: DualPE enables large DNA editing in tomato.
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Fig. 7: Pipeline and potential application of DualPE for editing large DNA fragments in plants.
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Data availability

DNA sequencing data have been deposited in the National Center for Biotechnology Information Sequence Read Archive database with the BioProject accession code PRJNA1192508. For data visualization, we used GraphPad Prism v.8.0.1, Microsoft Excel 2021, PowerPoint 2021 and Adobe Illustrator 2020. Source data are provided with this paper.

Code availability

The source code for DualPE-Finder is available at https://github.com/ZongyuanLab/DualPE. An interactive web page for designing dual-pegRNA for DualPE is available at http://wheat.cau.edu.cn/DualPE_Finder/.

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Acknowledgements

We thank C. Gao (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for suggestions on the manuscript. This work was supported by grants from the National Key Research and Development Program of China (no. 2021YFF1000800 to Y. Zong, no. 2022YFF1002801 to Y.W. and no. 2023YFD1202904 to L.C.), the Biological Breeding-Major Projects (no. 2023ZD0407403 to Y.W.), the National Natural Science Foundation of China (no. 32270429 to Y. Zong and no. 32122051 to Y.W.) and the Chinese Universities Scientific Fund (no. 2024TC162 to Y. Zong).

Author information

Author notes

  1. These authors contributed equally: Yidi Zhao, Zhengwei Huang, Ximeng Zhou, Wan Teng.

Authors and Affiliations

  1. Frontiers Science Center for Molecular Design Breeding (MOE), Key Laboratory of Crop Heterosis and Utilization (MOE) and Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing, China

    Yidi Zhao, Zhengwei Huang, Ximeng Zhou, Zehua Liu, Wenping Wang, Jing Liu, Wenxi Wang, Lingling Chai, Weilong Guo, Jie Liu, Zhongfu Ni, Qixin Sun & Yuan Zong

  2. Center for Genome Editing, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    Wan Teng, Shengjia Tang & Yanpeng Wang

  3. College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China

    Shengjia Tang & Yanpeng Wang

  4. College of Horticulture, China Agricultural University, Beijing, China

    Ying Liu & Na Zhang

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Contributions

Y. Zong, Y.W. and Y. Zhao designed the project. Y. Zhao, Z.H., X.Z., W.T., Z.L., Wenping Wang, S.T. and Y.L. performed the experiments. Y. Zong, Y.W. and Y. Zhao wrote the manuscript. Jing Liu, Wenxi Wang, L.C., N.Z., W.G., Jie Liu, Z.N. and Q.S. contributed to the discussion and revised the manuscript. Y. Zong and Y.W. supervised the project.

Corresponding authors

Correspondence to Yanpeng Wang or Yuan Zong.

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Competing interests

Y. Zong, Y.W., Q.S., Z.N. and Y. Zhao have submitted a provisional patent application (Chinese patent application no. 2024117368334) based on the results reported in this paper through China Agricultural University. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 dPCR results for detection of large DNA deletion in wheat protoplasts.

a, Scheme of the dPCR assay for quantification of deletion events. b-h, Digital fluorescence level in dPCR assay for 507-bp deletion (b), 1.5-kb deletion (c), 2.3-kb deletion (d), 4.8-kb deletion (e), 79.3-kb deletion (f), 258.0-kb deletion (g) and 365.9-kb deletion (h).

Extended Data Fig. 2 Sanger sequencing chromatograms of deletions induced by Cas9, WT-DualPE and DualPE in wheat protoplasts.

a-g, Represented Sanger sequencing chromatograms for 507-bp deletion (a), 1.5-kb deletion (b), 2.3-kb deletion (c), 4.8-kb deletion (d), 79.3-kb deletion (e), 258.0-kb deletion (f) and 369.5-kb deletion (g). Red arrows represent the junction of two cut/nick sites.

Extended Data Fig. 3 Identification and analysis of the wheat mutants for 365.9-kb deletion.

a, Schematic representation of the expression vector pB-DualPE for Agrobacterium transformation. b,c, Gel electrophoresis of PCR products for 365.9-kb deletion in T1 generation of T0-8 (b) and T0-13 (c) line. The 428-bp band is the desired size for the deletion, while the 603-bp band is the expected size for the wild-type sequence. WT, wild-type control plant. d, Segregation analysis of T0-8 and T0-13 lines derived from DualPE-mediated 365.9-kb deletions of wheat plants. HO, homozygous; HE, heterozygous; WT, wild-type.

Source data

Extended Data Fig. 4 Mutation type of byproducts for WT-DualPE and DualPE to generate replacement in wheat protoplasts.

a, Schematic diagram of three types of outcomes for replacement. The resulted outcomes for replacement can be divided into three types. (1) accurate replacement, (2) imperfect replacement, and (3) direct deletion. b,c, Represented byproducts which evaluated by high-throughput amplicon sequencing for 60-bp deletion with 38-bp insertion (b), and 4.8-kb deletion with 34-bp insertion (c).

Extended Data Fig. 5 Identification and analysis of the replacement in wheat mutants at the site of VRT-A2.

a, Schematic representation of the replacement of 567-bp sequence with 157 bp induced by DualPE. Spacers are shown in orange and green for pegL and pegR, respectively. RT templates encoding insertions marked as 3’ flap are shown in purple and blue, and blue and pink for pegL and pegR, respectively. Homologous overlap between pegL and pegR is shown in blue. The original 560 bp sequences are highlighted in blue, and the additional 7 bp flanking sequences that were deleted for PAM design are highlighted in red. The desired 157 bp insertion sequences are shown in purple, blue, and pink. b, Sanger sequencing chromatograms of T0-2, T0-8, T0-12, and T0-14. c, Segregation analysis of T0-2 and T0-8 lines derived from DualPE-mediated replacement. HO, homozygous; HE, heterozygous; WT, wild type.

Extended Data Fig. 6 Gel electrophoresis of PCR products and Sanger sequencing chromatograms of inversion in wheat protoplasts.

a, Amplification of target genomic region using inversion-specific primers amplifying either junction-L or junction-R. The inversion amplicons are denoted with an red arrow. An untreated protoplasts sample served as control. b-f, Sanger sequencing chromatograms of inversions induced by Cas9, WT-DualPE and DualPE at junction-L and- junction-R for sized 713-bp inversion (b), 1.5-kb inversion (c), 2.6-kb inversion (d), 74.4-kb inversion (e) and 252.6-kb inversion (f). Red arrows represent the junction of left or right.

Source data

Extended Data Fig. 7 Identification and analysis of the inversion for wheat mutants of Fragment 20-22.

a, Representative agarose gel of PCR products for genotyping of the 7.4-kb inversion. The 752-bp band and the 606-bp band are the desired bands for the left and right junction of the inversion, respectively, while the 598-bp and the 760-bp bands are the expected size for the wild-type sequence. WT, wild-type. T0-1, T0-2, T0-3, T0-4, T0-5, T0-7, T0-8, T0-9, T0-10, and T0-11 were homozygous. b, Representative Sanger sequencing chromatograms of the 7.4-kb inversion mediated by DualPE in wheat plants. T0-2 was identified as the precise inversion mutant at both Junction-L and Junction-R. In contrast, T0-5 contained a single nucleotide polymorphism (SNP) at the intended Junction-R, likely resulting from byproducts associated with the pegRNA scaffold. Meanwhile, T0-28 exhibited insertions and deletions (indels) at the targeted Junction-L, which can be attributed to the presence of micro-homologous sequences. c, Representative agarose gel of PCR products for genotyping of the 19.2-kb inversion. The 325-bp band and the 730-bp band are the desired bands for the left and right junction of the inversion, respectively, while the 695-bp and the 360-bp bands are the expected size for the wild-type sequence. WT, wild-type. T0-7 is homozygous. d, Representative Sanger sequencing chromatograms of the 19.2-kb inversion mediated by DualPE in wheat plants. T0-7 was identified as the precise inversion mutant at both Junction-L and Junction-R. In contrast, T0-10 contained a single nucleotide polymorphism (SNP) at the intended Junction-L, likely resulting from byproducts associated with the pegRNA scaffold. e, Representative Sanger sequencing chromatograms of the precise 82.8-kb inversion mediated by DualPE in wheat plants (T0-42).

Source data

Extended Data Fig. 8 Sanger sequencing chromatograms of large DNA editing induced by DualPE in N. benthamiana and tomato cells.

a-c, Sanger sequencing chromatograms of precise deletions induced by DualPE in N. benthamiana for sized 1.6-kb deletion (a), 5.3-kb deletion (b), and 134.7-kb deletion (c). d,e, Sanger sequencing chromatograms of replacements induced by DualPE in N. benthamiana for replacement of 106 bp with 34 bp (d), and replacement of 1.6 kb with 66 bp (e). The repetitive sequence of the 3xFlag was highlighted in red. f-j, Sanger sequencing chromatograms of precise inversions at junction-L and junction-R induced by DualPE in N. benthamiana for sized 829-bp inversion (f), 3.6-kb inversion (g), 3.9-kb inversion (h), 4.2-kb inversion (i), and 20.1-kb inversion (j). k,l, Sanger sequencing chromatograms of precise edits induced by DualPE in tomato for 725-bp deletion (k), 1.1-kb inversion (l). The red arrows represent the junction of two nick sites.

Extended Data Fig. 9 Identification and analysis of DualPE-mediated large DNA mutants in tomato plants.

a, Representative agarose gel of PCR products for genotyping of the 725-bp deletion. The 201-bp band is the desired size for the deletion, while the 926-bp band is the expected size for the wild-type sequence. T0-3, T0-9, T0-11 and T0-18 were homozygous. b, Sanger sequencing chromatograms of the precise 725-bp deletion. The left protospacer and right protospacer are shown in red and blue, respectively. c, Representative agarose gel of PCR products for genotyping of the 725-bp inversion. The 378-bp band and the 330-bp band are the desired bands for the left and right junction of the inversion, respectively, while the 353-bp and the 355-bp bands are the expected size for the wild-type sequence. d, Sanger sequencing chromatograms of the precise 725-bp inversion. The left protospacer, right protospacer and inverted sequences are shown in red, blue and purple, respectively. e, Representative agarose gel of PCR products for genotyping of the 1.1-kb replacement with 38 bp. The 469-bp band is the desired size for the replacement, while the 1572-bp band is the size for the wild-type sequence. WT, wild-type control plant. T0-3, T0-5 and T0-8 were homozygous. f, Sanger sequencing chromatograms of the precise 1.1-kb replacement with 38 bp. The desired replacements are shown in brown.

Source data

Extended Data Fig. 10 Input and output page of the dual-pegRNA designing webserver.

a, The input page of DualPE-Finder webserver includes the input textbox, user-defined parameters and primer design. b, Output page of DualPE-Finder webserver. The webserver displays all possible candidate pegLs and pegRs, providing details on spacer, PBS, RTT, primers and sequence after editing. Candidates are recommended based on the cumulative distance of pegL nicking from the start position and pegR nicking from the end position of the desired editing fragments, specifically ranked from the shortest to the longest distance.

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Unprocessed gels for Extended Data Fig. 9a,c,e.

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Zhao, Y., Huang, Z., Zhou, X. et al. Precise deletion, replacement and inversion of large DNA fragments in plants using dual prime editing. Nat. Plants 11, 191–205 (2025). https://doi.org/10.1038/s41477-024-01898-3

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