Efficient generation of transgene-free canker-resistant Citrus sinensis using Cas12a/crRNA ribonucleoprotein

Citrus is one of the top three tree crops in the world and its production faces many devastating disease challenges such as citrus canker caused by Xanthomonas citri subsp. citri (Xcc). Genetic improvement of citrus via traditional approaches is a lengthy (approximately 20 years) and laborious process. Biotechnological approaches including CRISPR genome editing technologies have shown promise. However, none of the citrus plants generated by biotechnological approaches have been commercialized, which primarily resulted from the transgenic nature of the genetically modified plants. Here, we successfully developed transgene-free canker-resistant Citrus sinensis lines in the T0 generation within 10 months through transformation of embryogenic citrus protoplasts with Cas12a/crRNA ribonucleoprotein (RNP) to edit the canker susceptibility gene CsLOB1 . Among the 39 regenerated lines, 38 are biallelic/homozygous mutants based on Sanger sequencing analysis of targeting sites and whole genome sequencing, demonstrating a 97.4% biallelic/homozygous mutation rate. The edited lines do not contain off-target mutations. The CsLOB1 edited C. sinensis lines demonstrate no differences from wild type plants except canker resistance. Importantly, the transgene-free canker-resistant C. sinensis lines have received regulatory approval by USDA APHIS. This study presents an efficient genome editing approach for citrus using Cas12a/crRNA RNP, which has a broad impact on genetic improvement of elite citrus varieties and potentially other tree crops and their genetic study. This study represents a breakthrough by generating the first transgene-free canker-resistant C. sinensis lines that provide a sustainable and efficient solution to control citrus canker. ### Competing Interest Statement N. W., H. S. and Y. C. W. filed a PCT patent application based on the results reported in this paper. All other authors declare no competing financial interests.