Auxin affects gene editing efficiency through regulating chromatin accessibility and plant regeneration process

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Genetics

Background:

• CRISPR/Cas9 technology is crucial for crop genetic enhancement.
• Optimizing gene editing efficiency is a key research objective.

Purpose of the Study:

• To investigate the impact of exogenous auxin on CRISPR/Cas9 gene editing efficiency in plants.
• To elucidate the mechanisms underlying auxin's effect on gene editing.
• To develop an improved culture strategy for gene editing in plants.

Main Methods:

• In vitro tissue culture with varying auxin concentrations.
• RNA-Sequencing (RNA-Seq) for gene expression analysis.
• Nuclei staining and Transposase-Accessible Chromatin using sequencing (ATAC-Seq) for chromatin structure analysis.
• Development and validation of a two-phase culture strategy.

Main Results:

• Elevated auxin levels significantly enhanced gene editing efficiency and the frequency of edited T₀ plants.
• Higher auxin promoted callus growth but delayed shoot initiation and slightly reduced regeneration.
• Auxin treatment led to a more relaxed chromatin structure, increasing DNA accessibility for Cas9.
• A two-phase culture strategy successfully balanced enhanced editing with shoot regeneration.

Conclusions:

• Exogenous auxin positively influences CRISPR/Cas9 gene editing efficiency by altering chromatin accessibility and providing extended editing time.
• The findings offer new insights into auxin's role in gene editing and provide a theoretical basis for future research.
• The developed two-phase culture method can accelerate the application of gene editing technologies in diverse plant species.