High-efficiency prime editing with optimized, paired pegRNAs in plants.

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Genome Editing

Background:

• Prime editing (PE) is a powerful genome editing technology.
• Current PE applications are often limited by suboptimal editing efficiency.

Purpose of the Study:

• To enhance prime editing efficiency in rice.
• To identify optimal conditions for prime editing guide RNA (pegRNA) design.

Main Methods:

• Investigated the impact of prime binding site melting temperature on PE efficiency.
• Developed and tested a dual-pegRNA strategy for simultaneous PE.
• Utilized a web application, PlantPegDesigner, for pegRNA design.

Main Results:

• Optimal PE performance in rice was achieved with a melting temperature of 30°C for prime binding sites.
• Using two pegRNAs encoding the same edit in trans significantly boosted PE efficiency.
• Combined approaches increased PE efficiency by 2.9-fold to 17.4-fold.

Conclusions:

• Optimized pegRNA design and dual-pegRNA strategies markedly improve PE efficiency in rice.
• The PlantPegDesigner tool facilitates the design of high-performance pegRNAs.
• These advancements hold promise for accelerating crop improvement through precise genome editing.