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

Transgenic Res. 2024 Apr;33(1-2):59-66. doi: 10.1007/s11248-024-00380-2. Epub 2024 Apr 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.

Keywords: CRISPR; Citrus; Genome editing; Multi-intron-containing Cas9 gene; Multiplex genome editing; gRNA-tRNA.

MeSH terms

  • CRISPR-Cas Systems / genetics
  • Citrus* / genetics
  • Gene Editing*
  • Introns
  • RNA, Guide, CRISPR-Cas Systems
  • RNA, Transfer / genetics


  • RNA, Guide, CRISPR-Cas Systems
  • RNA, Transfer