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Non-transgenic gene editing of Citrus sinensis using CRISPR/Cas9 ribonucleoprotein complexes

Yuanchun Wang: CREC, university of florida

<div>Title: Non-transgenic gene editing of <em>Citrus sinensis</em> using CRISPR/Cas9 ribonucleoprotein complexes</p> <p>Yuanchun Wang, Hang Su, Shuming Wang, Tirtha Lamichhane, Nian Wang</p> <p>Citrus Research and Education Center, IFAS, University of Florida, Lake Alfred, FL</p> <p>YW and HS contributed equally to this work.</p> <p>*Corresponding author: Nian Wang, email:<;/p> <p>Abstract:</p> <p>Citrus is one of the most important tree crops in the world. However, citrus production is facing many challenges including many biotic and abiotic stresses. Among them, both citrus canker caused by <em>Xanthomonas citri</em> and Huanglongbing caused by <em>Candidatus</em> Liberibacter asiaticus cause severe economic losses to citrus production. Breeding disease-resistant varieties is the most efficient and sustainable approach to controlling most plant diseases. Traditional breeding to generate disease-resistant citrus varieties has been hindered by polyembryony, pollen-ovule sterility, sexual and graft incompatibilities, and extended juvenility. CRISPR technology has shown promise to speed up the breeding process for citrus. Here, we describe our current progress in generating transgene-free citrus using the CRISPR/Cas9 ribonucleoproteins (RNPs) method. The purified Cas9 shows nuclease activity at the presence of sgRNA. Mutation of <em>CsPDS</em> was observed. No off-targets were detected.</div>