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Transgene-free genome editing of vegetatively propagated and perennial plant species in the T0 generation via a co-editing strategy

Nature Plants volume 9, pages 1591–1597 (2023)Cite this article

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Abstract

Transgene-free plant genome editing in the T0 generation is highly desirable but challenging1,2. Here we achieved such a goal using a co-editing strategy via Agrobacterium-mediated transient expression of cytosine base editor to edit ALS encoding acetolactate synthase to confer herbicide chlorsulfuron resistance as a selection marker, Cas12a/CRISPR RNA for editing gene(s) of interest, and green fluorescent protein for selecting transgene-free transformants. The biallelic/homozygous transgene-free mutation rates for target genes among herbicide-resistant transformants ranged from 1.9% to 42.1% in tomato, tobacco, potato and citrus. This co-editing strategy is particularly useful for transgene-free genome editing of vegetatively propagated and perennial plant species in the T0 generation.

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Fig. 1: Transgene-free gene editing in the first generation (T0) in tomato.
Fig. 2: Transgene-free gene editing in the first generation (T0) in pummelo (C. maxima).

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Data availability

The raw reads of genome resequencing for pummelo plants were deposited in the NCBI Bioproject database under accession number PRJNA931434. The reference genome of pummelo was downloaded from public citrus genome database CPBD: Citrus Pan-genome to Breeding Database (http://citrus.hzau.edu.cn/index.php). The raw reads of genome resequencing for tomato plants were deposited in the NCBI Bioproject database under accession number PRJNA931572. The reference genome of tomato was downloaded from public tomato genome database of International Tomato Genome Sequencing Project https://solgenomics.net/organism/Solanum_lycopersicum/genome). Source data are provided with this paper.

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Acknowledgements

We thank Wang lab members for constructive suggestions and insightful discussions. This project was supported by funding from Florida Citrus Initiative Program, Citrus Research and Development Foundation, US Department of Agriculture National Institute of Food and Agriculture grants 2022-70029-38471, 2021-67013-34588, 2018-70016-27412 and 2016-70016-24833, FDACS Specialty Crop Block Grant Program, and Hatch project (FLA-CRC-005979) (N.W.).

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Author notes

  1. These authors contributed equally: Xiaoen Huang, Hongge Jia.

Authors and Affiliations

  1. Citrus Research and Education Center, Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Lake Alfred, FL, USA

    Xiaoen Huang, Hongge Jia, Jin Xu, Yuanchun Wang & Nian Wang

  2. Citrus Research and Education Center, Department of Plant Pathology, Institute of Food and Agricultural Sciences, University of Florida, Lake Alfred, FL, USA

    Jiawen Wen

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Contributions

X.H., H.J. and N.W. conceptualized and designed the experiments. X.H., H.J. and Y.W. performed the experiments. J.X. and J.W. performed bioinformatics. X.H., H.J. and N.W. wrote the manuscript with input from all co-authors.

Corresponding author

Correspondence to Nian Wang.

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Competing interests

N.W., H.J. and X.H. filed a PCT patent application based on the results reported in this paper. All other authors declare no competing financial interests.

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Nature Plants thanks Myeong-Je Cho, Xiaoou Dong, Pengcheng Wei, Lanqin Xia, 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 Generation of transgene-free, gene-edited plants in the first generation using the CBE-Cas12a-GFP construct.

The CBE-Cas12a-GFP construct consists of CBE, Cas12a, and GFP, each driven by its own promoter. CBE and its gRNA are used to base edit the ALS gene to confer resistance to herbicide chlorsulfuron; Cas12a and its crRNA are used for target gene editing; GPF is used for selecting transgene-free transformants. The construct is introduced into plants through Agrobacterium-mediated transient expression. Transformants are screened on media containing chlorsulfuron. Putative transgene-free, gene-edited plants are kept if they lack green fluorescence. Plants with fluorescence are discarded. Regenerants without GFP are subjected to genotyping and further analysis.

Extended Data Fig. 2 Establishment of herbicide-assisted transgene-free genome editing system.

a, The CBE-Cas12a-GFP-SlALS1 construct used in the generation of transgene-free, SlALS1-edited tomato. The gRNA for SlALS1 is boxed. The targeted nucleotides (CC) are highlighted in yellow. CsVMV, Cassava vein mosaic virus promoter; U6, citrus U6 promoter; CmYLCV, Cestrum yellow leaf curling virus promoter; CBE, cytosine base editor; T, terminator. For GFP, Nos terminator; for SlALS gRNA, poly (T) terminator; for CBE and Cas12a, HSP 18.2 terminator. RB, T-DNA right border; LB, T-DNA left border. b, PCR amplification of GFP in the regenerated chlorsulfuron-resistant tomato lines with or without green fluorescence. c, SlALS1 gene genotyping of chlorsulfuron-resistant tomato regenerants through restriction enzyme digestion of PCR amplicons with StyI. PCR amplicons spanning the SlALS1 gRNA region were subjected to restriction enzyme digestion with StyI. Editing of the targeted nucleotides abolishes the StyI recognition site, resulting in resistance to StyI digestion. Bottom text: SlALS1 genotypes in the edited lines were confirmed by Sanger sequencing. b and c were repeated two times independently with similar results. Source data are provided as a Source data file.

Source data

Extended Data Fig. 3 Tomato calli and shoots selected on media containing chlorsulfuron herbicide.

a, Tomato calli were grown on the medium containing 110 nM chlorsulfuron. b, Close-up view of calli from the selection medium shown in a. c, Tomato calli and shoots selected on medium containing 110 nM chlorsulfuron.

Extended Data Fig. 4 Heritability test.

a, SlER genotypes of the progenies of sler-4. Seeds of the sler-4 line were germinated, and the seedlings were genotyped at SlER. b, PCR amplification of GFP and Cas12a for lines listed in a. At least 16 clones for each gene from each plant were subjected to Sanger sequencing. This experiment was repeated two times independently with similar results. Source data are provided as a Source data file.

Source data

Extended Data Fig. 5 Green fluorescence observation of seeds of transgenic and transgene-free lines.

Detection of green fluorescence in sler-4 and transgenic control seeds was conducted under a GFP filter.

Extended Data Fig. 6 Efficient transgene-free gene editing of tomato in the T0 generation with 2 crRNAs.

a, Construct scheme showing 1 crRNA targeting SlRbohD. b, Construct scheme showing 2 crRNAs targeting SlRbohD. c, The SlRbohD genotypes of the transgene-free, homozygous/biallelic edited lines without green fluorescence. d, Comparison of rate of transgene-free, homozygous/biallelic mutants using 1 crRNA and 2 crRNAs. e, PCR amplification of GFP and Cas12a in the lines shown in c. This experiment was repeated two times independently with similar results. Source data are provided as a Source data file.

Source data

Extended Data Fig. 7 Transgene-free, multiplex gene editing of tomato in the first generation.

a, Generation of transgene-free, biallelic/homozygous double mutants of tomato for SlEDS1 and SlPAD4. b, PCR amplification of GFP and Cas12a from the edited sleds1/slpad4 mutant lines from a. This experiment was repeated two times independently with similar results. Source data are provided as a Source data file. c, Generation of transgene-free, biallelic/homozygous double mutants for SlDMR6 and SlINVINH1. d, PCR amplification of GFP and Cas12a from the edited sldmr6/slinvinh1 mutant lines from c. This experiment was repeated two times independently with similar results. Source data are provided as a Source data file.

Source data

Extended Data Fig. 8 Transgene-free gene editing in the first generation (T0) in tobacco and potato.

a-c, Co-editing of NtALS and NtPDS in Nicotiana tabacum. a, Albino phenotype with or without green fluorescence. Regenerants were selected on herbicide chlorsulfuron-containing media. Upper: transgenic albino tobacco plant; lower: transgene-free albino tobacco plant. b, Confirmation of transgene-free gene editing. PCR amplification of GFP and Cas12a in WT, non-transgenic (NT), and transgenic (T) plants. This experiment was repeated two times independently with similar results. Source data are provided as a Source data file. c, Genotypes of NtALS, NtPDS genes in a transgene-free, albino tobacco line from a. d & e, Transgene-free gene editing in potato. d, PCR amplification of GFP and Cas12a from a regenerated potato line 9 and control transgenic plant. This experiment was repeated two times independently with similar results. Source data are provided as a Source data file. e, Genotype of line 9 at StDMR6. crRNAs are underlined. 1 crRNA was used for StDMR6 editing.

Source data

Extended Data Fig. 9 Analysis of line ep-8.

a, Identification of the construct integration site in the tomato genome through whole genome sequencing. Blue, tomato DNA sequence; red, construct DNA sequence. b Illustrations demonstrating the partial integration of the transformation construct into the tomato genome. Arrows indicate the primers used in the following PCR confirmation. c, PCR confirmation. This experiment was repeated two times independently with similar results. Source data are provided as a Source data file.

Source data

Extended Data Table 1 Summary of the whole genome sequencing data
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Supplementary information

Supplementary Information

Supplementary Tables 1–5, Figs. 1–18 and Files 1 and 2.

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Huang, X., Jia, H., Xu, J. et al. Transgene-free genome editing of vegetatively propagated and perennial plant species in the T0 generation via a co-editing strategy. Nat. Plants 9, 1591–1597 (2023). https://doi.org/10.1038/s41477-023-01520-y

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