Citrus Research and Education Center, Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Lake Alfred, FL, USA.
Citrus Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Lake Alfred, FL, USA.
Nat Commun. 2023 Jul 5;14(1):3957. doi: 10.1038/s41467-023-39714-9.
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.
由柑橘溃疡病菌（Xanthomonas citri subsp. citri，Xcc）引起的柑橘溃疡病是一种世界性的破坏性柑橘病害。培育抗病品种是控制病害最有效、最环保和最经济的方法。然而，柑橘传统的育种方法耗时耗力。在这里，我们通过 Cas12a/crRNA 核糖核蛋白转化胚胎原生质体，在 T0 代中在 10 个月内开发出无转基因的抗溃疡柑橘品种。在 39 个再生系中，有 38 个是双等位基因/纯合突变体，表明双等位基因/纯合突变率为 97.4%。在编辑系中未检测到脱靶突变。CsLOB1 编辑系的溃疡抗性既表现在消除溃疡症状，又表现在抑制 Xcc 生长。无转基因的抗溃疡柑橘品种已获得美国农业部动植物卫生检验局（APHIS）的监管批准，无需接受美国环保署（EPA）的监管。本研究为柑橘溃疡病的防治提供了一种可持续且高效的解决方案，并为柑橘和其他作物提供了一种高效的无转基因基因组编辑策略。
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