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

Prime editors (PEs), which can install desired base edits without donor DNA or double-strand breaks, have been used in plants and can, in principle, accelerate crop improvement and breeding. However, their editing efficiency in plants is generally low. Optimizing the prime editing guide RNA (pegRNA) by designing the sequence on the basis of melting temperature, using dual-pegRNAs and engineering PEs have all been shown to enhance PE efficiency. In addition, an automated pegRNA design platform, PlantPegDesigner, has been developed on the basis of rice prime editing experimental data. In this protocol, we present detailed protocols for designing and optimizing pegRNAs using PlantPegDesigner, constructing engineered plant PE vectors with enhanced editing efficiency for prime editing, evaluating prime editing efficiencies using a reporter system and comparing the effectiveness and byproducts of PEs by deep amplicon sequencing. Using this protocol, researchers can construct optimized pegRNAs for prime editing in 4-7 d and obtain prime-edited rice or wheat plants within 3 months. This protocol uses PlantPegDesigner to design and optimize prime editing guide RNA and engineered plant prime editor vectors for efficient prime editing in monocot plants.

Automated summary

Prime editors (PEs) have been used to accelerate crop improvement and breeding in plants by installing desired base edits. However, the editing efficiency of PEs in plants is generally low. In this protocol, methods such as optimizing prime editing guide RNA (pegRNA), using dual-pegRNAs, and engineering PEs have been shown to enhance editing efficiency. A design platform, PlantPegDesigner, based on rice prime editing experimental data, has also been developed. This protocol provides detailed steps for designing and optimizing pegRNAs using PlantPegDesigner, constructing engineered plant PE vectors, evaluating editing efficiencies, and comparing the effectiveness and byproducts of PEs.