Genome Editing in Cotton Using CRISPR/Cas9 System.

Area of Science:

• Plant Biotechnology
• Genome Editing
• Molecular Biology

Background:

• The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is a powerful tool for precise genome modification.
• CRISPR/Cas9 technology has been increasingly applied for crop genetic improvement due to its efficiency and versatility.

Purpose of the Study:

• To present a simple and high-efficiency method for creating targeted gene mutations in the allotetraploid cotton genome using the CRISPR/Cas9 system.
• To demonstrate the application of this method using GhMYB25-like A and GhMYB25-like D genes as examples.

Main Methods:

• Utilized the CRISPR/Cas9 system for targeted mutagenesis in upland cotton (Gossypium hirsutum).
• Employed GhMYB25-like A and GhMYB25-like D genes, derived from the A and D subgenomes, as targets for mutation induction.
• Analyzed mutation frequencies and types through PCR product sequencing.

Main Results:

• Induced fragment truncation events in GhMYB25-like A and GhMYB25-like D genome sites at frequencies of 14.2-21.4%.
• Achieved high mutation frequencies at target DNA sites: 100% for GhMYB25-like A and 98.8% for GhMYB25-like D.
• Detected no off-target mutations in transgenic plants, even with a single nucleotide mismatch at a putative off-target site.

Conclusions:

• The developed CRISPR/Cas9 method is effective for generating targeted gene mutations in the cotton genome.
• This approach offers high efficiency and specificity, making it a valuable tool for cotton genetic improvement.
• The absence of off-target mutations suggests the reliability of this method for precise genome editing in cotton.