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An engineered prime editor with enhanced editing efficiency in plants

Nature Biotechnology volume 40, pages 1394–1402 (2022)Cite this article

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An Author Correction to this article was published on 08 April 2022

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Abstract

Prime editing is a versatile genome-editing technology, but it suffers from low editing efficiency. In the present study, we introduce optimized prime editors with substantially improved editing efficiency. We engineered the Moloney–murine leukemia virus reverse transcriptase by removing its ribonuclease H domain and incorporated a viral nucleocapsid protein with nucleic acid chaperone activity. Each modification independently improved prime editing efficiency by ~1.8–3.4-fold in plant cells. When combined in our engineered plant prime editor (ePPE), the two modifications synergistically enhanced the efficiency of base substitutions, deletions and insertions at various endogenous sites by on average 5.8-fold compared with the original PPE in cell culture. No significant increase in byproducts or off-target editing was observed. We used the ePPE to generate rice plants tolerant to sulfonylurea and imidazolinone herbicides, observing an editing frequency of 11.3% compared with 2.1% using PPE. We also combined ePPE with the previously reported dual-prime editing guide (peg) RNAs and engineered pegRNAs to further increase efficiency.

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Fig. 1: Improvement of prime editing efficiency by removing the RT RNase H domain and an addition of a viral NC protein in plant cells.
Fig. 2: Engineered prime editors for precise genome editing in plant cells.
Fig. 3: Effect of prime editors on off-target prime editing.
Fig. 4: Engineered prime editors generated mutations in plants.
Fig. 5: Prime editing efficiency is enhanced by combining the engineered prime editors with optimized pegRNAs in plant cells.

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Data availability

All data supporting the findings of the present study are available in the article, extended data and supplementary figures and tables, or are available from the corresponding author on request. The deep sequencing data have been deposited in an NCBI BioProject database (accession no. PRJNA802997). The Zhonghua11 genome is available at NCBI BioProject database (accession no. PRJNA602608). Plasmids encoding ePPE, ePPE–SpG and pH-ePPE are available from Addgene (plasmids 183095, 183096, 183097). Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (no. 31788103) and the Strategic Priority Research Program of the Chinese Academy of Sciences (nos. XDA24020102 to C.G, XDB27030201 to X.C. and XDA24020310 to Y.W.).

Author information

Author notes

  1. These authors contributed equally: Yuan Zong, Yijing Liu, Chenxiao Xue.

Authors and 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

    Yuan Zong, Yijing Liu, Chenxiao Xue, Boshu Li, Yanpeng Wang, Guanwen Liu & Caixia Gao

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

    Yijing Liu, Chenxiao Xue, Boshu Li, Ji Li, Xiaofeng Cao & Caixia Gao

  3. School of Life Science and Technology, ShanghaiTech University, Shanghai, China

    Xiangyang Li & Xingxu Huang

  4. State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    Ji Li & Xiaofeng Cao

  5. Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Beijing, China

    Xiaofeng Cao

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Contributions

Y.Z., Y.L., C.X., X.H. and C.G. designed the project. Y.Z., Y.L., C.X., B.L., X.L., J.L. and G.L. performed the experiments. Y.W. prepared the figures. Y.Z., Y.L., C.X., X.C. and C.G. wrote the manuscript. C.G. supervised the project.

Corresponding authors

Correspondence to Xiaofeng Cao or Caixia Gao.

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Nature Biotechnology thanks Hyongbum Henry Kim and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Prime editing induced by PPE, PPE-F155Y, PPE-F155V, PPE-F156Y, PPE-N200C and PPE-D524N.

(a) Frequencies of prime editing induced by PPE, PPE-F155Y, PPE-F155V, PPE-F156Y, PPE-N200C, PPE-D524N at six rice target sites. (b) The average editing frequencies induced by PPE, PPE-F155Y, PPE-F155V, PPE-F156Y, PPE-N200C and PPE-D524N across six targets. Frequencies (mean ± s.e.m.) were calculated using the data in a. P values were obtained using two-tailed Student’s t-tests. *P < 0.05.

Extended Data Fig. 2 Product purity for PPE, PPE-∆RNase H, PPE-NC-v1 and PPE-NC-v2.

Frequencies of prime editing and undesired byproducts induced by PPE, PPE-∆RNase H, PPE-NC-v1 and PPE-NC-v2 at 16 endogenous sites in rice protoplasts (a) and six target sites in wheat protoplasts (b). Fold-change in the observed prime editing edit:byproduct ratio for rice target sites (c), and for wheat targets (d). Values were calculated from the data presented in Fig. 1f and 1g respectively. Data and error bars reflect the mean and standard deviation of three independent biological replicates. Frequencies (means ± s.e.m.) were calculated from three independent experiments (n = 3).

Extended Data Fig. 3 Product purity for PPE, PPE-∆RNase H, PPE-NC-v1, and ePPE.

(a) Product purity in prime editing by PPE, PPE-∆RNase H, PPE-NC-v1, and ePPE at 12 endogenous sites in rice protoplasts. Frequencies (means ± s.e.m.) were calculated from three independent experiments (n = 3). (b) Fold-change in the observed prime editing edit:byproduct ratio for 12 rice target sites. Values were calculated from the data presented in Fig. 2c. Data and error bars reflect the mean and standard deviation of three independent biological replicates. Frequencies (means ± s.e.m.) were calculated from three independent experiments (n = 3).

Extended Data Fig. 4 Overall editing frequencies induced by PPE and ePPE.

The overall editing frequencies induced by PPE and ePPE at 12 target sites in Fig. 2e (a) and at 32 target sites in Fig. 2c,e,f (b). The average of editing frequencies using ePPE for each target were normalized to 1, and the frequencies using PPE for each target were adjusted accordingly (n = 3 independent experiments). P values were obtained using the two-tailed Student’s t-test. ****P < 0.0001.

Extended Data Fig. 5 Prime editing induced by PPE-SpG and ePPE-SpG in rice protoplasts.

(a) Schematic representation of PPE-SpG and ePPE-SpG. (b) Frequencies of prime editing induced by PPE-SpG and ePPE-SpG at four target sites. Frequencies (mean ± s.e.m.) were calculated from three independent experiments (n = 3). P values were obtained using two-tailed Student’s t-tests. *P < 0.05.

Extended Data Fig. 6 Comparison of prime editing and base editing.

The total editing efficiency (a), and the precise C>T or A>G editing efficiency without bystander edits (b) at the seven targets induced by prime editors and base editors. Frequencies (mean ± s.e.m.) were calculated from three independent experiments (n = 3).

Extended Data Fig. 7 Genotypes of prime-edited OsALS-T6 rice mutants.

(a) Schematic representation of pH-ePPE. (b) The results of PCR-RE assays analyzing 12 representative OsALS-T6 plantlets (T0-1 to T0-12). restriction enzyme. ‘M’ represents marker. ‘WT/D’ represents digested PCR products of wild-type. ‘WT/U’ represents undigested PCR products of wild-type (Untreated). Arrowheads indicate the bands anticipated from BsrDI restriction enzyme. (c) Sanger sequencing chromatograms of representative seven prime-edited heterozygous and five chimera mutants. Red arrows represent the desired edits. One biological experiment was performed.

Extended Data Fig. 8 Comparison of the prime editing efficiency induced by PPE or ePPE with NGG-pegRNA, CCN-pegRNA and dual-pegRNA strategies.

(a) Frequencies of prime editing induced by PPE and ePPE at six rice target sites using NGG-pegRNA, CCN-pegRNA and dual-pegRNA strategies. The edits were referred to the base on the DNA forward strand. (b) Overall editing frequencies induced by PPE and ePPE containing NGG-pegRNA, CCN-pegRNA and dual-pegRNA. The average editing frequencies using ePPE-dual-pegRNA for each target were normalized to 1, and the frequencies using others for each target were adjusted accordingly. (c) Product purity in prime editing by PPE and ePPE using NGG-pegRNA, CCN-pegRNA and dual-pegRNA strategies. Data and error bars reflect the mean and standard deviation of three independent biological replicates. Frequencies (means ± s.e.m.) were calculated from three independent experiments (n = 3). P values were obtained using two-tailed Student’s t-tests. **P < 0.01, ****P < 0.0001.

Extended Data Fig. 9 Product purity induced by different PPEs and different engineered pegRNA forms.

(a) Product purity in prime editing by different PPEs and different engineered pegRNA forms at seven endogenous sites in rice protoplasts. Frequencies (means ± s.e.m.) were calculated from three independent experiments (n = 3). (b) Fold-change in the observed prime editing edit:byproduct ratio for seven rice target sites. Values were calculated from the data presented in Fig. 5a. Data and error bars reflect the mean and standard deviation of three independent biological replicates. Frequencies (means ± s.e.m.) were calculated from three independent experiments (n = 3).

Extended Data Fig. 10 Product purity induced by different PPEs and different pegRNA forms.

(a) Product purity in prime editing by different PPEs and different pegRNA forms at seven endogenous sites in rice protoplasts. Frequencies (means ± s.e.m.) were calculated from three independent experiments (n = 3). (b) Fold-change in the observed prime editing edit:byproduct ratio for seven rice target sites. Values were calculated from the data presented in Fig. 5d. Data and error bars reflect the mean and standard deviation of three independent biological replicates. Frequencies (means ± s.e.m.) were calculated from three independent experiments (n = 3).

Supplementary information

Supplementary Information

Supplementary Figs. 1–3, Tables 1–7, Notes 1 and 2 and Sequences.

Reporting Summary

Supplementary Data 1

The flow cytometry of BFP-to-GFP conversion induced by different PPEs.

Source data

Source Data Fig. 1

Unprocessed agarose gel.

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Zong, Y., Liu, Y., Xue, C. et al. An engineered prime editor with enhanced editing efficiency in plants. Nat Biotechnol 40, 1394–1402 (2022). https://doi.org/10.1038/s41587-022-01254-w

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