Multiplexed gene editing with a multi-intron containing Cas9 gene in citrus

The citrus industry holds significant economic importance in Florida, being one of the leading producers of oranges and grapefruits in the United States. However, several diseases, such as canker and huanglongbing along with natural disasters like hurricanes have rigorously affected citrus production, quality, and yield. Improving citrus through traditional breeding methods requires significant challenges due to time constraints and complexity in genetic enhancements. To overcome these limitations, 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 ( PDS ) 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 HLB. Competing interests The authors declare that they have no competing interests. Supplementary Information Supplementary File 1 ### Competing Interest Statement The authors have declared no competing interest.