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

Abstract

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.

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Fig. 1: The effects on PE in rice protoplasts of varying Tm-directed PBS length and forms of pegRNA.
Fig. 2: Overview of prime editing in rice and wheat.
Fig. 3: Experimental validation of the PlantPegDesigner web application.
Fig. 4: Construction of vectors for plant PEs.
Fig. 5: Input and output pages of PlantPegDesigner.

Data availability

All the data shown in this protocol are deposited in NCBI BioProject under accession codes PRJNA605069, PRJNA605074, and PRJNA702010. An example dataset for amplicon deep sequencing analysis used in this protocol is available on GitHub at https://github.com/ReiGao/GEanalysis.

Code availability

All the code used in this protocol is available on GitHub at https://github.com/ReiGao/GEanalysis. The PlantPegDesigner web application code is available at https://github.com/JinShuai001/PlantPegDesigner.

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Acknowledgements

We thank K. T. Zhao for his insightful comments on the manuscript. This work was supported by the Ministry of Agriculture and Rural Affairs of China, the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA24020102 to C.G.), the Young Elite Scientists Sponsorship Program of the China Association for Science and Technology (2020QNRC001 to S.J.) and the Postdoctoral Innovative Talent Support Program of China (BX2021353 to Q.L.).

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C.G. S.J., Q.L. and Q.G. wrote the manuscript; Q.L. and S.J. designed figures; C.G. supervised the project;

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Correspondence to Caixia Gao.

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Nature Protocols thanks Goetz Hensel, Yiping Qi and Seiichi Toki for their contribution to the peer review of this work.

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Key references using this protocol

Lin, Q. et al. Nat. Biotechnol. 38, 582–585 (2020): https://doi.org/10.1038/s41587-020-0455-x

Lin, Q. et al. Nat. Biotechnol. 39, 923–927 (2021): https://doi.org/10.1038/s41587-021-00868-w

Zong, Y. et al. Nat. Biotechnol. 40, 1394–1402 (2022): https://doi.org/10.1038/s41587-022-01254-w

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Jin, S., Lin, Q., Gao, Q. et al. Optimized prime editing in monocot plants using PlantPegDesigner and engineered plant prime editors (ePPEs). Nat Protoc (2022). https://doi.org/10.1038/s41596-022-00773-9

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