Double step screening using endogenous marker improves relative gene targeting efficiency in Arabidopsis

Area of Science:

• Plant biotechnology
• Molecular biology
• Genomics

Background:

• Gene targeting (GT) enables precise manipulation of plant genomes but suffers from low efficiency, particularly in seed plants.
• Existing CRISPR/Cas systems have not sufficiently boosted GT efficiency for routine use in plant research.
• Developing efficient GT methods is crucial for advancing plant genetic engineering.

Purpose of the Study:

• To establish a novel surrogate screening method for enhancing CRISPR/Cas9-mediated gene targeting efficiency in Arabidopsis.
• To demonstrate the effectiveness of simultaneous mutagenesis of the MAR1 gene for screening GT events.
• To assess the potential for broad application of this surrogate system in plant GT.

Main Methods:

• Implementation of a CRISPR/Cas9 system for gene targeting in Arabidopsis.
• Introduction of simultaneous mutagenesis of the endogenous MAR1 gene to confer kanamycin resistance.
• Development of a double-step screening strategy to identify successful GT events.
• Analysis of the precision and heritability of the targeted genetic modifications.

Main Results:

• The developed surrogate screening method significantly improved the relative efficiency of CRISPR/Cas9-mediated GT in Arabidopsis.
• Simultaneous mutagenesis of the MAR1 gene served as an effective selectable marker for GT events.
• The double-step screening strategy resulted in up to a four-fold increase in GT efficiency.
• Precise and heritable GT events were efficiently screened at multiple endogenous sites.

Conclusions:

• The reported surrogate screening method offers a substantial improvement in gene targeting efficiency in Arabidopsis.
• This approach facilitates the efficient screening of precise and heritable GT events.
• The principle of this surrogate system holds potential for widespread application in plant gene editing.