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Prime genome editing in rice and wheat

Nature Biotechnology volume 38, pages 582–585 (2020)Cite this article

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Abstract

Prime editors, which are CRISPR–Cas9 nickase (H840A)–reverse transcriptase fusions programmed with prime editing guide RNAs (pegRNAs), can edit bases in mammalian cells without donor DNA or double-strand breaks. We adapted prime editors for use in plants through codon, promoter, and editing-condition optimization. The resulting suite of plant prime editors enable point mutations, insertions and deletions in rice and wheat protoplasts. Regenerated prime-edited rice plants were obtained at frequencies of up to 21.8%.

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Fig. 1: Frequency of prime editing in rice and wheat protoplasts.
Fig. 2: Optimized prime editors for precise genome editing in rice.

Data availability

All data supporting the findings of this study are available in the article or in Supplementary information files, or are available from the corresponding author upon request. In terms of sequence data, the rice LOC_Os identifiers (http://rice.plantbiology.msu.edu/) are LOC_Os01g55540 (OsAAT), LOC_Os03g54790 (OsALS), LOC_Os03g05730 (OsCDC48), LOC_Os09g26999 (OsDEP1), LOC_Os06g04280 (OsEPSPS), LOC_Os08g03290 (OsGAPDH). The NCBI GenBank identifiers are KJ697755 (TaGW2), KF009556 (TaMLO), KJ000052 (TaGASR7), JF683316 (TaDME), GU167921 (TaLOX2), FJ459808 (TaUbi10). The deep sequencing data have been deposited in an NCBI BioProject database (accession codes PRJNA605069 and PRJNA605074). Plasmids encoding nCas9-PPE, QPM-sgR (for nicking sgRNA construction), and pH-nCas9-PPE (for pH-nCas9-PPE2/3/3b construction) will be made available through Addgene.

References

  1. Voytas, D. F. & Gao, C. Precision genome engineering and agriculture: opportunities and regulatory challenges. PLoS Biol. 12, e1001877 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Wang, W. et al. Genomic variation in 3,010 diverse accessions of Asian cultivated rice. Nature 557, 43–49 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Puchta, H. & Fauser, F. Gene targeting in plants: 25 years later. Int. J. Dev. Biol. 57, 629–637 (2013).

    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. 29, 667–697 (2019).

    Article  Google Scholar 

  5. Svitashev, S. et al. Targeted mutagenesis, precise gene editing, and site-specific gene insertion in maize using Cas9 and guide RNA. Plant Physiol. 169, 931–945 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  6. 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 

  7. Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, 1248–1256 (2016).

    Article  CAS  Google Scholar 

  9. Gaudelli, N. M. et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Kim, Y. B. et al. Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nat. Biotechnol. 35, 371–376 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 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 

  12. Plant, A. L., Covey, S. N. & Grierson, D. Detection of a subgenomic mRNA for gene V, the putative reverse transcriptase gene of cauliflower mosaic virus. Nucleic Acids Res. 13, 8305–8321 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Lim, D. & Maas, W. K. Reverse transcriptase-dependent synthesis of a covalently linked, branched DNA-RNA compound in E. coli B. Cell 56, 891–904 (1989).

    Article  CAS  PubMed  Google Scholar 

  14. Zong, Y. et al. Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Nat. Biotechnol. 35, 438–440 (2017).

    Article  CAS  PubMed  Google Scholar 

  15. Gao, Y. & Zhao, Y. Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. J. Integr. Plant Biol. 56, 343–349 (2014).

    Article  CAS  PubMed  Google Scholar 

  16. 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 

  17. 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 

  18. Wang, Y. et al. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat. Biotechnol. 32, 947–951 (2014).

    Article  CAS  PubMed  Google Scholar 

  19. Xing, H. et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 14, 327 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Čermák, T. et al. A multipurpose toolkit to enable advanced genome engineering in plants. Plant Cell 29, 1196–1217 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  21. 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 

  22. 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 

Download references

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (31788103), the Strategic Priority Research Program of the Chinese Academy of Sciences (Precision Seed Design and Breeding, XDA24000000), and the National Key Research and Development Program of China (2016YFD0101804). A.V.A, A.R., J.L.D., and D.R.L. were supported by US National Institutes of Health (NIH) grants U01AI142756, RM1HG009490, R01EB022376, R35GM118062, and by the Howard Hughes Medical Institute (HHMI).

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Author notes

  1. These authors contributed equally: Qiupeng Lin, Yuan Zong, 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

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

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

    Qiupeng Lin, Chenxiao Xue, Shuai Jin, Zixu Zhu, Yanpeng Wang & Caixia Gao

  3. Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA

    Andrew V. Anzalone, Aditya Raguram, Jordan L. Doman & David R. Liu

  4. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    Andrew V. Anzalone, Aditya Raguram, Jordan L. Doman & David R. Liu

  5. Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA

    Andrew V. Anzalone, Aditya Raguram, Jordan L. Doman & David R. Liu

Authors
  1. Qiupeng Lin
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Contributions

Q.L., Y.Z., C.X., Y.W., A.V.A., A.R, J.L.D, D.R.L., and C.G. designed the project; Q.L., Y.Z., C.X., S.W., S.J., and Z.Z. performed the experiments; Q.L., A.V.A., A.R, J.L.D, D.R.L., and C.G. wrote the manuscript.

Corresponding author

Correspondence to Caixia Gao.

Ethics declarations

Competing interests

The authors have submitted a patent application based on the results reported in this paper. D.R.L. is a consultant for and co-founder of Beam Therapeutics, Prime Medicine, Pairwise Plants, and Editas Medicine—companies that use genome editing.

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Supplementary information

Supplementary Figures

Supplementary Figures 1–8, Supplementary Tables 1–7, Supplementary Sequences 1–3, and Supplementary Data 1.

Reporting Summary

Unprocessed gels for Supplementary Fig. 8b

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Lin, Q., Zong, Y., Xue, C. et al. Prime genome editing in rice and wheat. Nat Biotechnol 38, 582–585 (2020). https://doi.org/10.1038/s41587-020-0455-x

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