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.

Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is an economically important disease in most citrus production regions worldwide. Xcc secretes a transcriptional activator like effector (TALE) PthA4 to bind to the effector binding elements (EBEs) in the promoter region of canker susceptibility gene LOB1 to activate its expression, which in turn causes canker symptoms. Editing the EBE region with Cas9/gRNA has been used to generate canker resistant citrus plants. However, most of the EBE-edited lines generated contain indels of 1–2 bp, which has higher possibility to be overcome by PthA4 adaptation. The adaptation capacity of TALEs inversely correlates with the number of mismatches with the EBE. LbCas12a/crRNA is known to generate longer deletion than Cas9. In this study, we used a temperature-tolerant and more efficient LbCas12a variant (ttLbCas12a), harboring the single substitution D156R, to modify the EBE region of LOB1. We first constructed GFP-p1380N-ttLbCas12a:LOBP, which was shown to be functional via Xcc-facilitated agroinfiltration in Pummelo (Citrus maxima) leaves. Subsequently, we stably expressed ttLbCas12a:LOBP in Pummelo. Eight transgenic lines were generated, with seven lines showing 100% mutations of the EBE, among which one line is homozygous. The EBE-edited lines had the ttLbCas12a-mediated deletions of up to 10 bp. Importantly, the seven lines were canker resistant and no off-targets were detected. In summary, ttLbCas12a can be used to efficiently generate biallelic/homozygous citrus mutant lines with short deletions, thus providing a useful tool for the functional study and breeding of citrus.