Generation of transgene-free canker-resistant Citrus sinensis cv. Hamlin in the T0 generation through Cas12a/CBE co-editing

Citrus canker disease affects citrus production. This disease is caused by subsp. citri (Xcc). Previous studies confirmed that during Xcc infection, PthA4, a transcriptional activator like effector (TALE), is translocated from the pathogen to host plant cells. PthA4 binds to the effector binding elements (EBEs) in the promoter region of canker susceptibility gene (EBE -LOBP) to activate its expression and subsequently cause canker symptoms. Previously, the Cas12a/CBE co-editing method was employed to disrupt EBE -LOBP of pummelo, which is highly homozygous. However, most commercial citrus cultivars are heterozygous hybrids and more difficult to generate homozygous/biallelic mutants. Here, we employed Cas12a/CBE co-editing method to edit EBE -LOBP of Hamlin ( ), a commercial heterozygous hybrid citrus cultivar grown worldwide. Binary vector GFP-p1380N-ttLbCas12a:LOBP1-mPBE:ALS2:ALS1 was constructed and shown to be functional via Xcc-facilitated agroinfiltration in Hamlin leaves. This construct allows the selection of transgene-free regenerants via GFP, edits to generate chlorsulfuron-resistant regenerants as a selection marker for genome editing resulting from transient expression of the T-DNA via nCas9-mPBE:ALS2:ALS1, and edits gene(s) of interest (i.e., EBE -LOBP in this study) through ttLbCas12a, thus creating transgene-free citrus. Totally, 77 plantlets were produced. Among them, 8 plantlets were transgenic plants (#Ham 1 - #Ham 8), 4 plantlets were transgene-free (#Ham 1 - #Ham 4), and the rest were wild type. Among 4 transgene-free plantlets, three lines (#Ham 1, #Ham 2 and #Ham 3) contained biallelic mutations in EBE , and one line (#Ham 4) had homozygous mutations in EBE . We achieved 5.2% transgene-free homozygous/biallelic mutation efficiency for EBE -LOBP in cv. Hamlin, compared to 1.9% mutation efficiency for pummelo in a previous study. Importantly, the four transgene-free plantlets and 3 transgenic plantlets that survived were resistant against citrus canker. Taken together, Cas12a/CBE co-editing method has been successfully used to generate transgene-free canker-resistant cv. Hamlin in the T0 generation via biallelic/homozygous editing of EBE of the canker susceptibility gene