Visual Assay for Gene Editing Using a CRISPR/Cas9 System in Carrot Cells

Area of Science:

• Plant biotechnology
• Molecular biology
• Genetics

Background:

• CRISPR/Cas9 technology has revolutionized genome editing across various species.
• Optimizing CRISPR/Cas9 methods is crucial for efficient gene editing applications.
• Model systems are valuable for demonstrating and validating gene editing tools.

Purpose of the Study:

• To establish and demonstrate a model carrot callus system for CRISPR/Cas9 targeted mutagenesis.
• To validate the efficiency of CRISPR/Cas9 vectors using this model system.
• To visually distinguish gene editing events in carrot cells.

Main Methods:

• Utilized carrot tissue accumulating anthocyanin pigments for visual distinction.
• Generated knockout mutations in the flavanone-3-hydroxylase (F3H) gene via CRISPR/Cas9.
• Employed restriction fragment analysis and sequencing to identify mutations (indels and deletions).

Main Results:

• Successfully demonstrated targeted mutagenesis in carrot callus using CRISPR/Cas9.
• F3H mutant cells showed a loss of anthocyanin pigment, appearing white.
• Identified both small indels and larger chromosomal deletions in targeted genes.

Conclusions:

• The carrot callus system provides a feasible and visually intuitive method for demonstrating CRISPR/Cas9 gene editing.
• This system can be effectively used for validating the efficiency of CRISPR/Cas9 vectors.
• The visual readout simplifies the identification of successful gene editing events in plants.