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An efficient DNA- and selectable-marker-free genome-editing system using zygotes in rice

Abstract

Technology involving the targeted mutagenesis of plants using programmable nucleases has been developing rapidly and has enormous potential in next-generation plant breeding. Notably, the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein-9 nuclease (Cas9) (CRISPR–Cas9) system has paved the way for the development of rapid and cost-effective procedures to create new mutant populations in plants1,2. Although genome-edited plants from multiple species have been produced successfully using a method in which a Cas9–guide RNA (gRNA) expression cassette and selectable marker are integrated into the genomic DNA by Agrobacterium tumefaciens-mediated transformation or particle bombardment3, CRISPR–Cas9 integration increases the chance of off-target modifications4, and foreign DNA sequences cause legislative concerns about genetically modified organisms5. Therefore, DNA-free genome editing has been developed, involving the delivery of preassembled Cas9–gRNA ribonucleoproteins (RNPs) into protoplasts derived from somatic tissues by polyethylene glycol–calcium (PEG–Ca2+)-mediated transfection in tobacco, Arabidopsis, lettuce, rice6, Petunia7, grapevine, apple8 and potato9, or into embryo cells by biolistic bombardment in maize10 and wheat11. However, the isolation and culture of protoplasts is not feasible in most plant species and the frequency of obtaining genome-edited plants through biolistic bombardment is relatively low. Here, we report a genome-editing system via direct delivery of Cas9–gRNA RNPs into plant zygotes. Cas9–gRNA RNPs were transfected into rice zygotes produced by in vitro fertilization of isolated gametes12 and the zygotes were cultured into mature plants in the absence of selection agents, resulting in the regeneration of rice plants with targeted mutations in around 14–64% of plants. This efficient plant-genome-editing system has enormous potential for the improvement of rice as well as other important crop species.

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Fig. 1: Genome editing in rice by direct delivery of CRISPR–Cas9 RNP into zygotes.
Fig. 2: Targeted mutagenesis of endogenous genes in rice by delivery of the CRISPR–Cas9 RNPs into zygotes.
Fig. 3: Genome editing in rice by direct delivery of the tRNA-based CRISPR–Cas9 multiplex vector into zygotes.

Data availability

Information on the multiplex vector (pMgPoef4_129-2A-GFP) has been deposited in GenBank with the accession number LC460477. The authors declare that all other data supporting the findings of this study are available in the paper and its supplementary information files or from the corresponding author upon reasonable request.

References

  1. 1.

    Belhaj, K., Chaparro-Garcia, A., Kamoun, S. & Nekrasov, V. Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods 9, 39 (2013).

    Article  Google Scholar 

  2. 2.

    Voytas, D. F. Plant genome engineering with sequence-specific nucleases. Annu. Rev. Plant Biol. 64, 327–350 (2013).

    CAS  Article  Google Scholar 

  3. 3.

    Kumar, V. & Jain, M. The CRISPR–Cas system for plant genome editing: advances and opportunities. J. Exp. Bot. 66, 47–57 (2015).

    CAS  Article  Google Scholar 

  4. 4.

    Lawrenson, T. et al. Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease. Genome Biol. 16, 258 (2015).

    Article  Google Scholar 

  5. 5.

    Jones, H. D. Regulatory uncertainty over genome editing. Nat. Plants 1, 14011 (2015).

    Article  Google Scholar 

  6. 6.

    Woo, J. W. et al. DNA-free genome editing in plants with preassembled CRISPR–Cas9 ribonucleoproteins. Nat. Biotechnol. 33, 1162–1164 (2015).

    CAS  Article  Google Scholar 

  7. 7.

    Subburaj, S. et al. Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins. Plant Cell Rep. 35, 1535–1544 (2016).

    CAS  Article  Google Scholar 

  8. 8.

    Malnoy, M. et al. DNA-free genetically edited grapevine and apple protoplast using CRISPR/Cas9 ribonucleoproteins. Front. Plant Sci. 7, 1904 (2016).

    Article  Google Scholar 

  9. 9.

    Andersson, M. et al. Genome editing in potato via CRISPR–Cas9 ribonucleoprotein delivery. Physiol. Plant. 164, 378–384 (2018).

    CAS  Article  Google Scholar 

  10. 10.

    Svitashev, S., Schwartz, C., Lenderts, B., Young, J. K. & Mark Cigan, A. Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes. Nat. Commun. 7, 13274 (2016).

    CAS  Article  Google Scholar 

  11. 11.

    Liang et al. Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nat. Commun. 8, 14261 (2017).

    CAS  Article  Google Scholar 

  12. 12.

    Uchiumi, T., Uemura, I. & Okamoto, T. Establishment of an in vitro fertilization system in rice (Oryza sativa L.). Planta 226, 581–589 (2007).

    CAS  Article  Google Scholar 

  13. 13.

    Hwang, W. Y. et al. Efficient genome editing in zebrafish using a CRISPR–Cas system. Nat. Biotechnol. 31, 227–229 (2013).

    CAS  Article  Google Scholar 

  14. 14.

    Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).

    CAS  Article  Google Scholar 

  15. 15.

    Russell, S. D. Double fertilization. Int. Rev. Cytol. 140, 357–388 (1992).

  16. 16.

    Raghavan, V. Some reflections on double fertilization, from its discovery to the present. New Phytol. 159, 565–583 (2003).

    CAS  Article  Google Scholar 

  17. 17.

    Koiso, N., Toda, E., Ichikawa, M., Kato, N. & Okamoto, T. Development of gene expression system in egg cells and zygotes isolated from rice and maize. Plant Direct 1, e00010 (2017).

    Article  Google Scholar 

  18. 18.

    Crossway, A. et al. Micromanipulation techniques in plant biotechnology. Biotechniques 4, 320–334 (1986).

    Google Scholar 

  19. 19.

    Davey, M. R., Cocking, E. C., Freeman, J., Pearce, N. & Tudor, I. Transformation of Petunia protoplasts by isolated Agrobacterium plasmids. Plant Sci. Lett. 18, 307–313 (1980).

    CAS  Article  Google Scholar 

  20. 20.

    Lurquin, P. F. Entrapment of plasmid DNA by liposomes and their interactions with plant protoplasts. Nucleic Acids Res. 6, 3773–3784 (1979).

    CAS  Article  Google Scholar 

  21. 21.

    Shillito, R. D., Saul, M. W., Paszkowski, J., Müller, M. & Potrykus, I. High efficiency direct gene transfer to plants. Nat. Biotechnol. 3, 1099–1103 (1985).

    Article  Google Scholar 

  22. 22.

    Kranz, E., von Wiegen, P. & Lörz, H. Early cytological events after induction of cell division in egg cells and zygote development following in vitro fertilization with angiosperm gametes. Plant J. 8, 9–23 (1995).

    Article  Google Scholar 

  23. 23.

    Toda, E., Ohnishi, Y. & Okamoto, T. Development of polyspermic rice zygotes. Plant Physiol. 171, 206–214 (2016).

    CAS  Article  Google Scholar 

  24. 24.

    Mikami, M., Toki, S. & Endo, M. Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice. Plant Mol. Biol. 88, 561–572 (2015).

    CAS  Article  Google Scholar 

  25. 25.

    Jensen, K. T. et al. Chromatin accessibility and guide sequence secondary structure affect CRISPR–Cas9 gene editing efficiency. FEBS Lett. 591, 1892–1901 (2017).

    CAS  Article  Google Scholar 

  26. 26.

    Kranz, E. & Lörz, H. In vitro fertilization with isolated, single gametes results in zygotic embryogenesis and fertile maize plants. Plant Cell 5, 739–746 (1993).

    Article  Google Scholar 

  27. 27.

    Kovács, M., Barnabás, B. & Kranz, E. Electro-fused isolated wheat (Triticum aestivum L.) gametes develop into multicellular structures. Plant Cell Rep. 15, 178–180 (1995).

    Article  Google Scholar 

  28. 28.

    Kumlehn, J., Lörz, H. & Kranz, E. Differentiation of isolated wheat zygotes into embryos and normal plants. Planta 205, 327–333 (1998).

    CAS  Article  Google Scholar 

  29. 29.

    Leduc, N. et al. Isolated maize zygotes mimic in vivo embryonic development and express microinjected genes when cultured in vitro. Dev. Biol. 177, 190–203 (1996).

    CAS  Article  Google Scholar 

  30. 30.

    Holm, P. B. et al. Regeneration of fertile barley plants from mechanically isolated protoplasts of the fertilized egg cell. Plant Cell 6, 531–543 (1994).

    CAS  Article  Google Scholar 

  31. 31.

    Uchiumi, T., Komatsu, S., Koshiba, T. & Okamoto, T. Isolation of gametes and central cells from Oryza sativa L. Sex Plant Reprod. 19, 37–45 (2006).

    Article  Google Scholar 

  32. 32.

    Toda, E., Ohnishi, Y. & Okamoto, T. Electro-fusion of gametes and subsequent culture of zygotes in rice. Bio-protocol 6, e2074 (2016).

    Article  Google Scholar 

  33. 33.

    Toki, S. et al. Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice. Plant J. 47, 969–976 (2006).

    CAS  Article  Google Scholar 

  34. 34.

    Osakabe, Y. et al. Optimization of CRISPR/Cas9 genome editing to modify abiotic stress responses in plants. Sci. Rep. 6, 26685 (2016).

    CAS  Article  Google Scholar 

  35. 35.

    Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415 (2003).

    CAS  Article  Google Scholar 

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Acknowledgements

We thank A. Ide (RIKEN Cluster for Science) for performing part of the molecular experiments, the RIKEN Bio Resource Center (Tsukuba) for providing cultured rice cells (Oc line), and the RIKEN CSRS Sequencing Service (Yokohama) for sequencing. This work was supported, in part, by the MEXT KAKENHI (Grant-in-Aid for Scientific Research on Innovative Areas, grant no. 17H05845 to T.O.) and JSPS KAKENHI (Grant-in-Aid for Challenging Exploratory Research, grant no. 16K14742 to T.O.). This work was supported by JST’s Program on Open Innovation Platform with Enterprises, Research Institute and Academia (OPERA) (Y.O.).

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E.T., T.K., N.Kato and T.O. designed the experiments; E.T. performed most of the experiments; N.Koiso provided technical assistance to E.T. and performed some parts of the experiments; A.T. performed some parts of the molecular experiments; M.I. prepared transformed rice plants; T.K. provided technical assistance; K.O. and Y.O. performed vector construction; H.S., N.Kato and T.O. supervised the project; and E.T. and T.O. conceived the project and wrote the article.

Corresponding authors

Correspondence to Erika Toda or Takashi Okamoto.

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Competing interests

T.O., N.Kato, E.T., N.Koiso, M.I. and T.K. are co-inventors on a patent application covering the results described in this paper.

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Supplementary Methods, Supplementary Figures 1–11, Supplementary Tables 1–10 and Supplementary References.

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Toda, E., Koiso, N., Takebayashi, A. et al. An efficient DNA- and selectable-marker-free genome-editing system using zygotes in rice. Nat. Plants 5, 363–368 (2019). https://doi.org/10.1038/s41477-019-0386-z

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