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A design optimized prime editor with expanded scope and capability in plants

Nature Plants volume 8, pages 45–52 (2022)Cite this article

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Abstract

The ability to manipulate the genome in a programmable manner has illuminated biology and shown promise in plant breeding. Prime editing, a versatile gene-editing approach that directly writes new genetic information into a specified DNA site without requiring double-strand DNA breaks, suffers from low efficiency in plants1,2,3,4,5. In this study, N-terminal reverse transcriptase–Cas9 nickase fusion performed better in rice than the commonly applied C-terminal fusion. In addition, introduction of multiple-nucleotide substitutions in the reverse transcriptase template stimulated prime editing with enhanced efficiency. By using these two methods synergistically, prime editing with an average editing frequency as high as 24.3% at 13 endogenous targets in rice transgenic plants, 6.2% at four targets in maize protoplasts and 12.5% in human cells was achieved, which is two- to threefold higher than the original editor, Prime Editor 3. Therefore, our optimized approach has potential to make more formerly non-editable target sites editable, and expands the scope and capabilities of prime editing in the future.

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Fig. 1: Optimized prime editors for precise genome editing in rice calli.
Fig. 2: Optimized prime editors for precise genome editing in rice transgenic plants.
Fig. 3: Optimized prime editors for precise genome editing in maize protoplast and a summary of editing results in plants.

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Data availability

All data supporting the findings of this study are available in the article or in Supplementary information files. The next-generation sequencing data for plant and HEK293T cells have been deposited in an NCBI BioProject database and the accession code is PRJNA658855.

Code availability

Analyses of prime-editing mutation frequency were performed using our own script, which can be found on https://github.com/yangyongxing1991/Deep-Seq-Analysis.git.

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Acknowledgements

This work was supported by Beijing Scholars Program (BSP041), National Key Research and Development Program of China (2019YFA0802800) and the 13th Five-Year National Key R & D Program of China (2017YFD0102000). We thank R. Chen, Biotechnology Research Institute, Tianjin Academy of Agricultural Sciences for writing the script of analysing the NGS data. We also thank R. McKenzie, Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

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Author notes

  1. These authors contributed equally: Wen Xu, Yongxing Yang.

Authors and Affiliations

  1. Beijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding, Beijing Academy of Agriculture & Forestry Sciences, Beijing, People’s Republic of China

    Wen Xu, Yongxing Yang, Si Zhao, Lu Zhang, Guiting Kang, Feipeng Wang, Hongmei Yi, Wen Ren, Lu Li, Xiaoqing He, Jiuran Zhao & Jinxiao Yang

  2. Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, Peking University Genome Editing Research Center, College of Life Sciences, Peking University, Beijing, People’s Republic of China

    Biying Yang, Chuanmao Zhang & Bo Zhang

  3. Department of Biomedical Engineering, College of Engineering, Peking University, Beijing, People’s Republic of China

    Christopher J. Krueger

  4. Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA

    Christopher J. Krueger

  5. College of Agronomy and Biotechnology, Southwest University, Chongqing, People’s Republic of China

    Qianlin Xiao

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  1. Wen Xu
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Contributions

J.Y. and J.Z. supervised the project. J.Y. and W.X. designed the experiments. Y.Y., Q.X., S.Z., L.Z., G.K., F.W., H.Y., W.R., L.L. and X.H. performed all the experiments in plants. B.Y. and C.J.K. performed all the experiments in human cells. B.Z. and C.Z. designed the experiment in human cells. W.X., Y.Y. and J.Y. wrote and modified the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Jiuran Zhao or Jinxiao Yang.

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Competing interests

The authors submitted patent applications based on the results reported in this paper.

Peer review information

Nature Plants thanks Yiping Qi and the other, anonymous, reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Optimized prime editors for precise genome editing in HEK293T cells.

a, Schematic representation of two PE vectors in HEK293T cells, cPE and nPE. b, Genomic sequences after prime editing using different RT-S and RT-M templates at the three endogenous targets, counting the first base 3’ of the pegRNA-induced nick as position +1. Bases desired to be edited are highlighted in red and the assistant bases are marked in blue.

Extended Data Fig. 2 Prime editing efficiency in HEK293T cells.

a, Prime editing efficiencies in HEK293T cells using four different strategies. Values and error bars reflect mean ± s.d. of n = 3 independent biological replicates. b, Fold of enhancement of multiple-nucleotide substitutions in the RT template on cPE and nPE.

Extended Data Fig. 3 Detailed editing alleles in HEK293T cells.

Detailed editing alleles edited with cPE-RT-S and cPE-RT-M strategies at the three targets in HEK293T cells was analyzed with CRISPResso2 after Hi-Tom sequencing. For each target, the reference sequence from this region is at the top. Negative control indicated a non-targeting pegRNA transfection. RT-S and RT-M strategies were indicated by the distance from the nick and the substituted bases. Edited bases in the cells were indicated with bold font. All alleles observed with frequency ≥1.5% are shown.

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Xu, W., Yang, Y., Yang, B. et al. A design optimized prime editor with expanded scope and capability in plants. Nat. Plants 8, 45–52 (2022). https://doi.org/10.1038/s41477-021-01043-4

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