Efficient and versatile multiplex prime editing in hexaploid wheat

Area of Science:

• Plant biotechnology
• Gene editing technologies
• Crop improvement

Background:

• Prime editing (PE) offers precise DNA modifications but faces efficiency limitations in plants.
• Existing prime editing systems require optimization for broad application in crop species like wheat.

Purpose of the Study:

• To develop an improved prime editing system for hexaploid wheat with enhanced efficiency.
• To establish a multiplex prime editing platform for simultaneous multi-gene editing in wheat.

Main Methods:

• Engineered an upgraded plant prime editor (ePPEplus) by introducing a V223A substitution into the reverse transcriptase of the ePPEmax architecture.
• Tested ePPEplus in hexaploid wheat protoplasts and regenerated plants for efficiency and multiplexing capability.

Main Results:

• ePPEplus demonstrated significantly higher editing efficiency, averaging 33.0-fold and 6.4-fold increases over previous systems.
• Achieved robust multiplex prime editing, simultaneously editing 4-10 genes in protoplasts and up to 8 genes in regenerated plants.
• Observed editing frequencies up to 74.5% in multiplex editing experiments.

Conclusions:

• The ePPEplus system substantially enhances prime editing efficiency and capability in hexaploid wheat.
• The developed multiplex prime editing platform is a powerful tool for stacking multiple agronomic traits in wheat.
• This advancement broadens the application of prime editing for accelerating crop breeding and genetic improvement.