Genome Editing in Cotton with the CRISPR/Cas9 System

Area of Science:

• Agricultural Science
• Molecular Biology
• Genetics

Background:

• CRISPR/Cas9 gene editing offers revolutionary potential for crop improvement, but predicting single guide RNA (sgRNA) efficiency is challenging.
• Ineffective sgRNAs in cotton (Gossypium hirsutum L.) lead to wasted resources due to labor-intensive transformation processes.
• Validated sgRNAs are crucial for efficient gene functional studies and developing improved crop varieties.

Purpose of the Study:

• To develop a fast and efficient transient expression system for validating sgRNA functionality in cotton.
• To analyze the types and efficiency of CRISPR/Cas9-induced mutations in key cotton genes.
• To demonstrate the feasibility of multiplex gene editing and targeted gene fragment deletion in cotton.

Main Methods:

• Developed a transient expression system for rapid sgRNA validation in cotton.
• Applied the system to validate sgRNAs targeting GhPDS, GhCLA1, and GhEF1 genes.
• Analyzed CRISPR/Cas9-induced mutations, including deletions, multiplex targeting, and gene fragment deletions.

Main Results:

• Deletions were the most frequent mutation type (approximately 64%) in cotton cotyledons.
• Demonstrated successful multiplex gene targeting of GhPDS and GhEF1 using multiple sgRNAs.
• Achieved high mutation efficiency (80.6%) in transgenic cotton targeting GhCLA1, resulting in an albino phenotype.

Conclusions:

• The developed transient assay is an effective tool for validating sgRNA functionality in cotton.
• CRISPR/Cas9 can efficiently induce mutations in homeologous cotton genes, essential for this allotetraploid species.
• The system facilitates targeted gene editing, including multiplexing and fragment deletion, accelerating cotton genetic improvement.