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Generation of the transgene-free canker-resistant Citrus sinensis using Cas12a/crRNA ribonucleoprotein in the T0 generation

Author

Listed:
  • Hang Su

    (University of Florida)

  • Yuanchun Wang

    (University of Florida)

  • Jin Xu

    (University of Florida)

  • Ahmad A. Omar

    (University of Florida
    Zagazig University)

  • Jude W. Grosser

    (University of Florida)

  • Milica Calovic

    (University of Florida)

  • Liyang Zhang

    (Integrated DNA Technologies, Inc)

  • Yu Feng

    (University of Florida)

  • Christopher A. Vakulskas

    (Integrated DNA Technologies, Inc)

  • Nian Wang

    (University of Florida)

Abstract

Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is a destructive citrus disease worldwide. Generating disease-resistant cultivars is the most effective, environmentally friendly and economic approach for disease control. However, citrus traditional breeding is lengthy and laborious. Here, we develop transgene-free canker-resistant Citrus sinensis lines in the T0 generation within 10 months through transformation of embryogenic protoplasts with Cas12a/crRNA ribonucleoprotein to edit the canker susceptibility gene CsLOB1. Among the 39 regenerated lines, 38 are biallelic/homozygous mutants, demonstrating a 97.4% biallelic/homozygous mutation rate. No off-target mutations are detected in the edited lines. Canker resistance of the cslob1-edited lines results from both abolishing canker symptoms and inhibiting Xcc growth. The transgene-free canker-resistant C. sinensis lines have received regulatory approval by USDA APHIS and are exempted from EPA regulation. This study provides a sustainable and efficient citrus canker control solution and presents an efficient transgene-free genome-editing strategy for citrus and other crops.

Suggested Citation

  • Hang Su & Yuanchun Wang & Jin Xu & Ahmad A. Omar & Jude W. Grosser & Milica Calovic & Liyang Zhang & Yu Feng & Christopher A. Vakulskas & Nian Wang, 2023. "Generation of the transgene-free canker-resistant Citrus sinensis using Cas12a/crRNA ribonucleoprotein in the T0 generation," Nature Communications, Nature, vol. 14(1), pages 1-12, December.
    • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-39714-9
    DOI: 10.1038/s41467-023-39714-9
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