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

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Genomics

Background:

• CRISPR/Cas9 technology enables gene editing for citrus disease management.
• Existing expression systems have limitations in efficiency across diverse citrus varieties.

Purpose of the Study:

• To develop and evaluate a novel multiplex gene editing system for citrus.
• To enhance the efficiency of targeting the phytoene desaturase gene in citrus.

Main Methods:

• Employed a unified vector with a multi-intron Cas9 gene and multiple gRNAs separated by tRNA sequences.
• Targeted the phytoene desaturase gene in 'Carrizo' citrange and 'Duncan' grapefruit.
• Utilized multiplex gene editing for simultaneous targeting of multiple gene sites.

Main Results:

• Achieved significantly boosted gene editing efficiency in both 'Carrizo' citrange and 'Duncan' grapefruit.
• Demonstrated successful mutations in all three targeted sites within the phytoene desaturase gene.
• The unified vector approach proved effective for multiplex genome editing in citrus.

Conclusions:

• The developed multiplex CRISPR/Cas9 system with a multi-intron Cas9 and gRNA-tRNA array offers potent tools for citrus genome editing.
• This approach shows promise for combating citrus diseases like canker and huanglongbing.
• Facilitates efficient genetic improvement in diverse citrus varieties.