Multiplexed gene editing in citrus by using a multi-intron containing Cas9 gene

• Published in: Transgenic research 2024-04, Vol.33 (1-2), p.59-66
• Main Authors: Sarkar, Poulami, Santiago Vazquez, Jorge, Zhou, Mingxi, Levy, Amit, Mou, Zhonglin, Orbović, Vladimir
• Format: Article
• Language: English
• Subjects: Animal Genetics and Genomics; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Brief Report; Citrus; Citrus fruits; CRISPR; CRISPR-Cas systems; Desaturase; Disease; Efficiency; Food quality; genes; Genetic Engineering; Genetically altered foods; genetically modified organisms; Genome editing; Genomes; grapefruits; greening disease; gRNA; Life Sciences; Molecular Medicine; Plant bacterial diseases; Plant Genetics and Genomics; Proteins; Transfer RNA; Transgenics; tRNA
• ISSN: 0962-8819; 1573-9368
• DOI: 10.1007/s11248-024-00380-2

Several expression systems have been developed in clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9) framework allowing for gene editing of disease-associated genes across diverse citrus varieties. In this study, we present a new approach employing a multi-intron containing Cas9 gene plus multiple gRNAs separated with tRNA sequences to target the phytoene desaturase gene in both ‘Carrizo’ citrange and ‘Duncan’ grapefruit. Notably, using this unified vector significantly boosted editing efficiency in both citrus varieties, showcasing mutations in all three designated targets. The implementation of this multiplex gene editing system with a multi-intron-containing Cas9 plus a gRNA-tRNA array demonstrates a promising avenue for efficient citrus genome editing, equipping us with potent tools in the ongoing battle against several diseases such as canker and huanglongbing.