An efficient multiplex approach to CRISPR/Cas9 gene editing in citrus

Area of Science:

• Plant biotechnology
• Genome engineering
• Molecular biology

Background:

• CRISPR/Cas9 gene editing efficiency is crucial for routine implementation, especially in difficult-to-transform species like Citrus.
• Simultaneous targeting of multiple genes (multiplex editing) is essential for genetic screening and complex genome engineering but poses significant challenges.

Purpose of the Study:

• To improve multiplex gene editing efficiency in Carrizo citrange (Citrus) using tRNA-based sgRNA arrays.
• To evaluate and optimize the efficacy of various promoters for Cas9 endonuclease and sgRNA expression in Citrus.
• To provide quantitative insights into CRISPR/Cas9 vector design for simultaneous multi-gene editing.

Main Methods:

• Utilized tRNA-based sgRNA arrays for multiplex gene editing in Carrizo citrange.
• Evaluated a range of promoters (e.g., UBQ10, RPS5a, Pol III, ES8Z from Arabidopsis) for driving Cas9 and sgRNA expression.
• Optimized the expression levels of both Cas9 endonuclease and sgRNA arrays.

Main Results:

• Achieved significant improvements in multiplex gene editing efficiency through promoter optimization.
• Identified UBQ10 or RPS5a promoters driving zCas9i as effective for high editing levels.
• Demonstrated that Pol III promoters, along with UBQ10 and ES8Z, are robust for sgRNA array expression, impacting multi-target editing efficiency.

Conclusions:

• This study offers quantitative insights into CRISPR/Cas9 vector design for enhanced multiplex gene editing in Citrus.
• The findings have practical applications for simultaneous multi-gene editing in Citrus and potentially other eudicot species.
• Optimized promoter selection is critical for maximizing CRISPR/Cas9 efficacy in complex plant genome engineering.