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An engineered prime editor with enhanced editing efficiency in plants

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.

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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References

  1. Gao, C. Genome engineering for crop improvement and future agriculture. Cell 184, 1621–1635 (2021).

    Article  CAS  PubMed  Google Scholar 

  2. Anzalone, A. V., Koblan, L. W. & Liu, D. R. Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors. Nat. Biotechnol. 38, 824–844 (2020).

    Article  CAS  PubMed  Google Scholar 

  3. Zhu, H., Li, C. & Gao, C. Applications of CRISPR–Cas in agriculture and plant biotechnology. Nat. Rev. Mol. Cell Biol. 21, 661–677 (2020).

    Article  CAS  PubMed  Google Scholar 

  4. Chen, K., Wang, Y., Zhang, R., Zhang, H. & Gao, C. CRISPR/Cas genome editing and precision plant breeding in agriculture. Annu. Rev. Plant. Biol. 70, 667–697 (2019).

    Article  CAS  PubMed  Google Scholar 

  5. Ran, Y., Liang, Z. & Gao, C. Current and future editing reagent delivery systems for plant genome editing. Sci. China Life Sci. 60, 490–505 (2017).

    Article  CAS  PubMed  Google Scholar 

  6. Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Newby, G. A. & Liu, D. R. In vivo somatic cell base editing and prime editing. Mol. Ther. 29, 3107–3124 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Bosch, J. A., Birchak, G. & Perrimon, N. Precise genome engineering in Drosophila using prime editing. Proc. Natl Acad. Sci. USA 118, e2021996118 (2021).

    Article  CAS  PubMed  Google Scholar 

  9. Lin, Q. et al. Prime genome editing in rice and wheat. Nat. Biotechnol. 38, 582–585 (2020).

    Article  CAS  PubMed  Google Scholar 

  10. Petri, K. et al. CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells. Nat. Biotechnol. 40, 189–193 (2022).

    Article  CAS  PubMed  Google Scholar 

  11. Qian, Y. et al. Efficient and precise generation of Tay–Sachs disease model in rabbit by prime editing system. Cell Discov. 7, 50 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Liu, Y. et al. Efficient generation of mouse models with the prime editing system. Cell Discov. 6, 27 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Jiang, Y. Y. et al. Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize. Genome Biol. 21, 257 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Lin, Q. et al. High-efficiency prime editing with optimized, paired pegRNAs in plants. Nat. Biotechnol. 39, 923–927 (2021).

    Article  CAS  PubMed  Google Scholar 

  15. Liu, Y. et al. Enhancing prime editing by Csy4-mediated processing of pegRNA. Cell Res. 31, 1134–1136 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Nelson, J. W. et al. Engineered pegRNAs improve prime editing efficiency. Nat. Biotechnol. https://doi.org/10.1038/s41587-021-01039-7 (2021).

  17. Das, D. & Georgiadis, M. M. The crystal structure of the monomeric reverse transcriptase from Moloney murine leukemia virus. Structure 12, 819–829 (2004).

    Article  CAS  PubMed  Google Scholar 

  18. Rein, A. Murine leukemia viruses: objects and organisms. Adv. Virol. 2011, 403419 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Lim, D. et al. Crystal structure of the moloney murine leukemia virus RNase H domain. J. Virol. 80, 8379–8389 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Gao, G., Orlova, M., Georgiadis, M. M., Hendrickson, W. A. & Goff, S. P. Conferring RNA polymerase activity to a DNA polymerase: a single residue in reverse transcriptase controls substrate selection. Proc. Natl Acad. Sci. USA 94, 407–411 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Boyer, P. L., Sarafianos, S. G., Arnold, E. & Hughes, S. H. Analysis of mutations at positions 115 and 116 in the dNTP binding site of HIV-1 reverse transcriptase. Proc. Natl Acad. Sci. USA 97, 3056–3061 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Katano, Y. et al. Generation of thermostable Moloney murine leukemia virus reverse transcriptase variants using site saturation mutagenesis library and cell-free protein expression system. Biosci. Biotechnol. Biochem. 81, 2339–2345 (2017).

    Article  CAS  PubMed  Google Scholar 

  23. Blain, S. W. & Goff, S. P. Effects on DNA synthesis and translocation caused by mutations in the RNase H domain of Moloney murine leukemia virus reverse transcriptase. J. Virol. 69, 4440–4452 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Herschhorn, A. & Hizi, A. Retroviral reverse transcriptases. Cell. Mol. Life Sci. 67, 2717–2747 (2010).

    Article  CAS  PubMed  Google Scholar 

  25. Katz, R. A. & Skalka, A. M. The retroviral enzymes. Annu. Rev. Biochem. 163, 133–173 (1994).

    Article  Google Scholar 

  26. Mougel, M., Houzet, L. & Darlix, J. L. When is it time for reverse transcription to start and go? Retrovirology 6, 24 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  27. Cannon, K., Qin, L., Schumann, G. & Boeke, J. D. Moloney murine leukemia virus protease expressed in bacteria is enzymatically active. Arch. Virol 143, 381–388 (1998).

    Article  CAS  PubMed  Google Scholar 

  28. Walton, R. T. et al. Unconstrained genome targeting with near-PAMless engineered CRISPR–Cas9 variants. Science 368, 290–296 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Jin, S. et al. Genome-wide specificity of prime editors in plants. Nat. Biotechnol. 39, 1292–1299 (2021).

    Article  CAS  PubMed  Google Scholar 

  30. Zong, Y. et al. Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A. Nat. Biotechnol. 36, 950–953 (2018).

    Article  CAS  Google Scholar 

  31. Richter, M. F. et al. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat. Biotechnol. 38, 883–891 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Powles, S. B. & Yu, Q. Evolution in action: plants resistant to herbicides. Annu. Rev. Plant Biol. 61, 317–347 (2010).

    Article  CAS  PubMed  Google Scholar 

  33. Chen, L. et al. Trp548Met mutation of acetolactate synthase in rice confers resistance to a broad spectrum of ALS-inhibiting herbicides. Crop J. 9, 750–758 (2021).

    Article  Google Scholar 

  34. Zheng, C. et al. A flexible split prime editor using truncated reverse transcriptase improves dual-AAV delivery in mouse liver. Mol. Ther. https://doi.org/10.1016/j.ymthe.2022.01.005 (2022).

  35. Song, M. et al. Generation of a more efficient prime editor 2 by addition of the Rad51 DNA-binding domain. Nat. Commun. 12, 5617 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Chen, P. J. et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184, 5635–5652.e5629 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Anzalone, A. V. et al. Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. Nat. Biotechnol. https://doi.org/10.1038/s41587-021-01133-w (2021).

  38. Li, C. et al. Expanded base editing in rice and wheat using a Cas9–adenosine deaminase fusion. Genome Biol. 19, 59 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Shan, Q. et al. Targeted genome modification of crop plants using a CRISPR–Cas system. Nat. Biotechnol. 31, 686–688 (2013).

    Article  CAS  PubMed  Google Scholar 

  40. Shan, Q. et al. Rapid and efficient gene modification in rice and Brachypodium using TALENs. Mol. Plant 6, 1365–1368 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Bae, S., Park, J. & Kim, J. S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Jin, S. et al. Rationally designed APOBEC3B cytosine base editors with improved specificity. Mol. Cell 79, 728–740 (2020).

    Article  CAS  PubMed  Google Scholar 

Download references

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

Authors and Affiliations

Authors

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

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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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