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

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. Development of canker-resistant citrus cultivars via traditional approaches is a lengthy and laborious process. Here, the authors report the generation of regulatory approval, transgene-free, canker-resistant sweet orange lines using Cas12a/crRNA ribonucleoprotein-based susceptibility gene editing strategy.

Automated summary

In this study, transgene-free canker-resistant Citrus sinensis lines were generated within 10 months through Cas12a/crRNA ribonucleoprotein-mediated editing of the CsLOB1 gene. These lines exhibited resistance to canker disease by suppressing canker symptoms and inhibiting the growth of Xcc. The development of these canker-resistant citrus cultivars provides a sustainable and efficient solution for canker control and presents a transgene-free genome editing strategy.