Optimized prime editing in monocot plants using PlantPegDesigner and engineered plant prime editors (ePPEs)

Area of Science:

• Plant biotechnology
• Genome editing technologies
• Crop improvement

Background:

• Prime editors (PEs) enable precise base edits without double-strand breaks or donor DNA, offering potential for accelerated crop breeding.
• Current PE efficiency in plants remains a significant limitation for widespread application.
• Previous strategies to enhance PE efficiency include pegRNA optimization, dual-pegRNA systems, and PE engineering.

Purpose of the Study:

• To provide detailed protocols for designing and optimizing prime editing guide RNAs (pegRNAs) for enhanced prime editing efficiency in plants.
• To present an automated pegRNA design platform, PlantPegDesigner, developed using rice experimental data.
• To detail methods for constructing engineered plant PE vectors, evaluating editing efficiency, and analyzing byproducts.

Main Methods:

• Utilized the automated PlantPegDesigner platform for pegRNA design and optimization based on melting temperature and other parameters.
• Constructed engineered plant prime editor (PE) vectors with improved editing capabilities.
• Employed a reporter system for evaluating prime editing efficiencies.
• Performed deep amplicon sequencing to compare PE effectiveness and identify byproducts.

Main Results:

• Developed and validated protocols for designing optimized pegRNAs within 4-7 days.
• Demonstrated the successful generation of prime-edited rice and wheat plants within 3 months.
• Established methods for robust evaluation of prime editing efficiency and byproduct analysis.

Conclusions:

• The presented protocols and tools significantly enhance prime editing efficiency in plants.
• This work facilitates the accelerated improvement and breeding of important crop species like rice and wheat.
• The optimized pegRNA design and vector construction methods provide a valuable resource for plant genome editing research.