CRISPR/Cas9-mediated editing of CsWRKY22 reduces susceptibility to Xanthomonas citri subsp. citri in Wanjincheng orange (Citrus sinensis (L.) Osbeck)

Key message CRISPR/Cas9-mediated editing of CsWRKY22 repressed canker development in Wanjincheng orange. Citrus canker, a destructive disease of citrus, is threatening the citrus industry worldwide. Breeding resistant cultivars is the most economical and effective approach to control citrus canker. Recently, CRISPR/Cas9-mediated genome editing was demonstrated to be a powerful tool for the improvement of citrus resistance to the disease. In our previous works, we confirmed that CsWRKY22 is involved in the plant immunity response to citrus canker in Wanjincheng orange (Citrus sinensis (L.) Osbeck). In this study, we targeted this gene to improve the resistance of Wanjincheng orange against citrus canker by CRISPR/Cas9-mediated editing. Sanger sequencing confirmed that CsWRKY22 in Wanjincheng orange contains CsWRKY22(G) and CsWRKY22 (c) alleles, and the ratio of CsWRKY22(G) to CsWRKY22 (c) is approximately 2:1. Four sgRNAs, which targeted the first exon of CsWRKY22, were selected for testing. In vitro cleavage activity analysis showed that two (W1 and W2) of the four sgRNAs displayed robust cleavage activities using PCR amplicons from the Wanjincheng orange genome as template. Subsequently, two constructs, pCas9/WRKY22sgRNA-W1 and pCas9/WRKY22sgRNA-W2, were used to modify the CsWRKY22. Three mutant plants were identified from seven independent transgenic plants. Based on Sanger sequencing, the W1-1, W2-2, and W2-3 mutant lines displayed 85.7%, 79.2%, and 68.2% mutation rates, respectively. Resistance evaluation indicated that the mutant plants showed decreased susceptibility to citrus canker. These results indicate that CRISPR/Cas9-targeted gene modification is an efficient approach for enhancing disease resistance in citrus.