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

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

Abstract

Prime editing (PE) applications are limited by low editing efficiency. Here we show that designing prime binding sites with a melting temperature of 30 °C leads to optimal performance in rice and that using two prime editing guide (peg) RNAs in trans encoding the same edits substantially enhances PE efficiency. Together, these approaches boost PE efficiency from 2.9-fold to 17.4-fold. Optimal pegRNAs or pegRNA pairs can be designed with our web application, PlantPegDesigner.

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Data availability

All data supporting the findings of this study are available in the article and its supplementary figures and tables or can be obtained from the corresponding authors upon reasonable request. For sequence data, rice locus identifiers (http://rice.plantbiology.msu.edu/) are as follows:

LOC_Os01g55540 (OsAAT), LOC_Os05g22940 (OsACC), LOC_Os03g54790 (OsALS), LOC_Os03g05730 (OsCDC48), LOC_Os09g26999 (OsDEP1), LOC_Os06g04280 (OsEPSPS), LOC_Os08g39890 (OsIPA1); LOC_Os10g40600 (OsNRT1.1B), LOC_Os08g03290 (OsGAPDH), LOC_Os03g08570 (OsPDS) and LOC_Os06g35970 (OsROC5). The deep sequencing data have been deposited in a National Center for Biotechnology Information BioProject database (accession code PRJNA702010). Plasmids pSpG-PPE, pOsU3, pTaU3 and pH-nCas9-PPE-V2 will be available through Addgene.

Code availability

The PlantPegDesigner web application code is available at GitHub (https://github.com/JinShuai001/PlantPegDesigner). The web portal server is accessible at http://www.plantgenomeediting.net for non-profit use.

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Acknowledgements

This work was supported by grants from the National Transgenic Science and Technology Program of China (2019ZX08010-001 and 2019ZX08010-003), the Strategic Priority Research Program of the Chinese Academy of Sciences (Precision Seed Design and Breeding, XDA24020310 and XDA24030504) and the National Natural Science Foundation of China (31788103 and 32001060).

Author information

Author notes

  1. These authors contributed equally: Qiupeng Lin, Shuai Jin, Yuan Zong, Hong Yu.

Affiliations

  1. State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China

    Qiupeng Lin, Shuai Jin, Yuan Zong, Zixu Zhu, Yanpeng Wang & Caixia Gao

  2. College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China

    Qiupeng Lin, Shuai Jin, Zixu Zhu, Guanwen Liu, Yanpeng Wang, Jiayang Li & Caixia Gao

  3. State Key Laboratory of Plant Genomics, and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Beijing, China

    Hong Yu, Liquan Kou & Jiayang Li

  4. State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

    Guanwen Liu & Jin-Long Qiu

  5. CAS Center for Excellence in Biotic Interactions, University of Chinese Academy of Sciences, Beijing, China

    Jin-Long Qiu

Contributions

Q.L., S.J., Y.Z., J.Q., J.L. and C.G. designed the project. Q.L., Y.Z., S.J., Z.Z., G.L. and Y.W. performed the experiments. H.Y., S.J. and L.K. developed the web application. Q.L., S.J., Y.Z., J.Q., J.L. and C.G. wrote the manuscript. J.L. and C.G. supervised the project.

Corresponding authors

Correspondence to
Jiayang Li or Caixia Gao.

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Competing interests

The authors have submitted a patent application based on the results reported in this paper.

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Peer review information Nature Biotechnology thanks the anonymous reviewers for their contribution to the peer review of this work.

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Lin, Q., Jin, S., Zong, Y. et al. High-efficiency prime editing with optimized, paired pegRNAs in plants.
Nat Biotechnol (2021). https://doi.org/10.1038/s41587-021-00868-w

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