Biallelic Editing of the LOB1 Promoter via CRISPR/Cas9 Creates Canker-Resistant 'Duncan' Grapefruit

Citrus canker caused by subsp. is one of the most devastating citrus diseases worldwide. Generating disease-resistant citrus varieties is considered one of the most efficient and environmentally friendly measures for controlling canker. subsp. causes canker symptoms by inducing the expression of canker susceptibility gene via PthA4, a transcription activator-like (TAL) effector, by binding to the effector binding element (EBE) in the promoter region. In previous studies, canker-resistant plants were generated by mutating the coding region or the EBE of . However, homozygous or biallelic canker-resistant plants have not been generated for commercial citrus varieties, such as grapefruit ( ), which usually contain two alleles of and thus, have two types of promoter sequences: TI LOBP and TII LOBP. Two different sgRNAs were used to target both EBE types. Both 35S promoter and Yao promoter were used to drive the expression of SpCas9p to modify EBE -LOBP in grapefruit. Using 'Duncan' grapefruit epicotyls as explants, 19 genome-edited grapefruit plants were generated with one biallelic mutant line (#Dun 7). subsp. caused canker symptoms on wild-type and nonbiallelic mutant plants but not on #Dun 7. XccPthA4 mutant containing the designer TAL effector dLOB1.5, which recognizes a conserved sequence in both wild-type and #Dun 7, caused canker symptoms on both wild-type and #Dun 7. No off-target mutations were detected in #Dun 7. This study represents the first time that CRISPR-mediated genome editing has been successfully used to generate disease-resistant plants for 'Duncan' grapefruit, paving the way for using disease-resistant varieties to control canker.