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


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.


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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.).

Author information




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).

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