Transgenerational CRISPR-Cas9 Activity Facilitates Multiplex Gene Editing in Allopolyploid Wheat

Area of Science:

• Agricultural Science
• Molecular Biology
• Genetics

Background:

• Multiplexed gene editing (MGE) enables simultaneous modification of multiple genomic loci in crops.
• CRISPR-Cas9 technology offers a precise tool for genetic trait improvement.

Purpose of the Study:

• To apply MGE using a tRNA-gRNA construct for simultaneous gene editing in hexaploid wheat.
• To investigate the effects of editing TaGW2, TaLpx-1, and TaMLO genes on agronomic traits.
• To assess the transgenerational activity of CRISPR-Cas9 editing.

Main Methods:

• Developed and utilized a multiplexed gene editing construct with tandemly arrayed tRNA-gRNA units.
• Applied the construct to hexaploid wheat to target TaGW2, TaLpx-1, and TaMLO genes.
• Analyzed heritable mutations and their effects on seed size and thousand grain weight across generations.

Main Results:

• Achieved knockout mutations in all homoeologous copies of the TaGW2 gene.
• Observed a significant increase in seed size and thousand grain weight due to TaGW2 editing.
• Demonstrated CRISPR-Cas9 editing activity in subsequent generations, indicating transgenerational inheritance of edits.

Conclusions:

• Transgenerational gene editing activity can be a source of novel genetic variation in crop progeny.
• CRISPR-Cas9-based MGE is effective for modifying multiple genes and enhancing agronomic traits in wheat.
• Cas9-inducible trait transfer through crossing offers a strategy for crop improvement.