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

Hang Su, Yuanchun Wang, Jin Xu, Ahmad A. Omar, Jude W. Grosser, Milica Calovic, Liyang Zhang, Yu Feng, Christopher A. Vakulskas and Nian Wang ()
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Hang Su: University of Florida
Yuanchun Wang: University of Florida
Jin Xu: University of Florida
Ahmad A. Omar: University of Florida
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

Nature Communications, 2023, vol. 14, issue 1, 1-12

Abstract: 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.

Date: 2023
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DOI: 10.1038/s41467-023-39714-9

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