Multiplexed promoter and gene editing in wheat using a virus-based guide RNA delivery system

Area of Science:

• Plant molecular biology
• Crop genetics
• Biotechnology

Background:

• CRISPR-Cas technology is crucial for crop improvement but faces limitations due to low genetic transformation efficiency in diverse cultivars.
• Virus-based delivery systems offer a promising solution to overcome these challenges for broad application.

Purpose of the Study:

• To develop and demonstrate an efficient virus-based CRISPR-Cas system for gene editing in wheat.
• To enable multiplexed editing of agronomic genes and create heritable deletions in regulatory regions.

Main Methods:

• Direct inoculation of wheat leaves with barley stripe mosaic virus (BSMV) transcripts carrying guide RNAs (sgRNA) into Cas9-expressing wheat.
• Marker-assisted introgression to transfer high-expressing Cas9 locus into adapted spring and winter wheat cultivars.
• Multiplexed editing of two agronomic genes using BSMV-sgRNAs.

Main Results:

• Successful generation of heritable precise deletions in the promoter region of a transcription factor.
• Demonstrated multiplexed editing of agronomic genes in adapted wheat cultivars.
• Validated the strategy for creating cis-regulatory diversity and large-scale gene editing.

Conclusions:

• The BSMV-mediated CRISPR-Cas delivery system is effective for precise and multiplexed gene editing in wheat.
• This strategy accelerates gene discovery and trait improvement by enabling efficient crop genome engineering.
• The approach is applicable to any adapted cultivar for targeted genetic modifications.