Efficient CRISPR/Cas9 genome editing with Citrus embryogenic cell cultures

Area of Science:

• Plant Biotechnology
• Genomics
• Molecular Biology

Background:

• Precise genome editing is crucial for plant research and crop improvement.
• Citrus embryogenic cell cultures offer a rapid method for generating edited plant lines.
• This study assessed genome editing capabilities in citrus cells.

Purpose of the Study:

• To evaluate the efficacy of two CRISPR/Cas9 constructs (pC-PDS1 and pC-PDS2) for genome editing in Citrus sinensis.
• To determine the efficiency of gene editing in citrus embryogenic cell cultures.
• To identify optimal strategies for successful genome editing in citrus.

Main Methods:

• Agrobacterium-mediated CRISPR/Cas9 system was employed.
• Two constructs with guide RNAs targeting the phytoene desaturase (PDS) gene were utilized.
• Citrus sinensis 'EV2' embryogenic cell cultures were used for transformation and analysis.

Main Results:

• Both pC-PDS1 and pC-PDS2 constructs successfully induced mutations in the PDS gene.
• All edited embryos exhibited an albino phenotype.
• Mutations included insertions, substitutions, and deletions near the protospacer adjacent motif.

Conclusions:

• Both tested CRISPR/Cas9 constructs are effective for genome editing in citrus embryogenic cell cultures.
• The efficiency of genome editing is significantly influenced by the choice of guide RNA.
• Embryogenic cell cultures provide a viable platform for advancing citrus genome editing techniques.