Cas12a/3 crRNAs RNP transformation enables transgene-free multiplex genome editing, long deletions, and inversions in citrus chromosome in the T0 generation
Cas12a/3 crRNAs RNP transformation enables transgene-free multiplex genome editing, long deletions, and inversions in citrus chromosome in the T0 generation
Abstract Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a devastating disease worldwide. Previously, we successfully generated canker-resistant Citrus sinensis cv. Hamlin lines in the T0 generation, achieving a mutation efficiency of 97.4%. This was achieved through the transformation of embryogenic protoplasts using the Cas12a/1 crRNA ribonucleoprotein (RNP) system to edit the canker susceptibility gene, CsLOB1, which led to small indels. Here, we transformed embryogenic protoplasts of Hamlin with Cas12a/3 crRNAs RNP, resulting in 100% efficiency in editing the CsLOB1 gene in the T0 generation. Among the 10 transgene-free genome-edited lines, long deletions were obtained in five lines. Additionally, inversions were observed in three of the five edited lines with long deletions, but not in any edited lines with short indel mutations, suggesting long deletions are required for inversions. Biallelic mutations were observed for each of the three target sites in 4 of the 10 edited lines when 3 crRNAs were used, demonstrating that transformation of embryogenic citrus protoplasts with Cas12a/3 crRNAs RNP can be very efficient for multiplex editing. Our analysis revealed the absence of off-target mutations in the edited lines. These cslob1 mutant lines were canker-resistant and no canker symptoms were observed after inoculation with Xcc and Xcc growth was significantly reduced in the cslob1 mutant lines compared to the wild type plants. Taken together, Cas12a/3 crRNAs RNP transformation of embryogenic protoplasts of citrus provides a promising solution for transgene-free multiplex genome editing with high efficiency and for deletion of long fragments.
Wang Yuanchun、Xu Jin、Omar Ahmad A.、Su Hang、Wang Nian、Grosser Jude W.
Citrus Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida||Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of FloridaCitrus Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida||Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of FloridaCitrus Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida||Department of Horticultural Sciences, Institute of Food and Agricultural Sciences, University of FloridaCitrus Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida||Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of FloridaCitrus Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida||Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of FloridaCitrus Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida||Department of Horticultural Sciences, Institute of Food and Agricultural Sciences, University of Florida
植物保护遗传学生物科学研究方法、生物科学研究技术
CRISPR/Cas12agenome editingnon-transgenicinversionXanthomonasCsLOB1disease resistance
Wang Yuanchun,Xu Jin,Omar Ahmad A.,Su Hang,Wang Nian,Grosser Jude W..Cas12a/3 crRNAs RNP transformation enables transgene-free multiplex genome editing, long deletions, and inversions in citrus chromosome in the T0 generation[EB/OL].(2025-03-28)[2025-05-04].https://www.biorxiv.org/content/10.1101/2024.06.13.598908.点此复制
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