AUTHOR=Jia Hongge , Omar Ahmad A. , Xu Jin , Dalmendray Javier , Wang Yuanchun , Feng Yu , Wang Wenting , Hu Zhuyuan , Grosser Jude W. , Wang Nian 

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

JOURNAL=Frontiers in Plant Science

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2024.1385768

DOI=10.3389/fpls.2024.1385768

ISSN=1664-462X

ABSTRACT=<p>Citrus canker disease affects citrus production. This disease is caused by <italic>Xanthomonas citri</italic> 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 <italic>LOB1</italic> (EBE<sub>PthA4</sub>-LOBP) to activate its expression and subsequently cause canker symptoms. Previously, the Cas12a/CBE co-editing method was employed to disrupt EBE<sub>PthA4</sub>-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<sub>PthA4</sub>-LOBP of Hamlin (<italic>Citrus sinensis</italic>), 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 <italic>ALS</italic> 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<sub>PthA4</sub>-LOBP in this study) through ttLbCas12a, thus creating transgene-free citrus. Totally, 77 plantlets were produced. Among them, 8 plantlets were transgenic plants (#Ham<sub>GFP</sub>1 - #Ham<sub>GFP</sub>8), 4 plantlets were transgene-free (#Ham<sub>NoGFP</sub>1 - #Ham<sub>NoGFP</sub>4), and the rest were wild type. Among 4 transgene-free plantlets, three lines (#Ham<sub>NoGFP</sub>1, #Ham<sub>NoGFP</sub>2 and #Ham<sub>NoGFP</sub>3) contained biallelic mutations in EBE<sub>pthA4</sub>, and one line (#Ham<sub>NoGFP</sub>4) had homozygous mutations in EBE<sub>pthA4</sub>. We achieved 5.2% transgene-free homozygous/biallelic mutation efficiency for EBE<sub>PthA4</sub>–LOBP in <italic>C. sinensis</italic> 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 <italic>C. sinensis</italic> cv. Hamlin in the T0 generation via biallelic/homozygous editing of EBE<sub>pthA4</sub> of the canker susceptibility gene <italic>LOB1.</italic></p>