Abstract

The CRISPR–Cas9 genome-editing tool and the availability of whole-genome sequences from plant species have revolutionized our ability to introduce targeted mutations into important crop plants, both to explore genetic changes and to introduce new functionalities. Here, we describe protocols adapting the CRISPR–Cas9 system to apple and grapevine plants, using both plasmid-mediated genome editing and the direct delivery of CRISPR–Cas9 ribonucleoproteins (RNPs) to achieve efficient DNA-free targeted mutations in apple and grapevine protoplasts. We provide a stepwise protocol for the design and transfer of CRISPR–Cas9 components to apple and grapevine protoplasts, followed by verification of highly efficient targeted mutagenesis, and regeneration of plants following the plasmid-mediated delivery of components. Our plasmid-mediated procedure and the direct delivery of CRISPR–Cas9 RNPs can both be utilized to modulate traits of interest with high accuracy and efficiency in apple and grapevine, and could be extended to other crop species. The complete protocol employing the direct delivery of CRISPR–Cas9 RNPs takes as little as 2–3 weeks, whereas the plasmid-mediated procedure takes >3 months to regenerate plants and study the mutations.

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Key references using this protocol

Malnoy, M. et al. Front. Plant Sci. 7, 1904 (2016): https://doi.org/10.3389/fpls.2016.01904

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Acknowledgements

This work was supported by the Council for Science, Technology and Innovation (CSTI), the Cross-ministerial Strategic Innovation Promotion Program (SIP), and the ‘Technologies for creating next-generation agriculture, forestry and fisheries’ program (funding agency: Bio-oriented Technology Research Advancement Institution, NARO) in Japan. This work was also supported by the Program on Open Innovation Platform with Enterprises, Research Institute and Academia (OPERA) in Japan and the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry (IPET) through the Agri-Bio Industry Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA, grant number: 116088-3).

Author information

Author notes

    • Riccardo Velasco

    Present address: Research Centre for Viticulture and Enology, CREA, Conegliano, TV, Italy

  1. These authors contributed equally: Yuriko Osakabe, Zhenchang Liang, Chidananda Nagamangala Kanchiswamy.

Affiliations

  1. Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima, Japan

    • Yuriko Osakabe
    •  & Keishi Osakabe
  2. Beijing Key Laboratory of Grape Sciences and Enology, Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing, China

    • Zhenchang Liang
    •  & Chong Ren
  3. Division of Fruit Breeding and Genetics, NARO Institute of Fruit Tree and Tea Science, Tsukuba, Ibaraki, Japan

    • Chikako Nishitani
  4. Division of Apple Research, NARO Institute of Fruit Tree and Tea Science, Morioka, Iwate, Japan

    • Masato Wada
  5. Faculty of Agriculture, Iwate University, Morioka, Iwate, Japan

    • Sadao Komori
  6. Research and Innovation Centre, Genomics and Biology of Fruit Crop Department, Fondazione Edmund Mach (FEM), San Michele all’Adige, TN, Italy

    • Mickael Malnoy
    • , Riccardo Velasco
    •  & Chidananda Nagamangala Kanchiswamy
  7. PLANTeDIT Pvt Ltd, Cork, Ireland

    • Michele Poli
    • , Ok-Jae Koo
    •  & Chidananda Nagamangala Kanchiswamy
  8. ToolGen, Seoul, South Korea

    • Min-Hee Jung
    •  & Ok-Jae Koo
  9. Italian Institute of Technology, Genova, Italy

    • Roberto Viola

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Contributions

Y.O., Z.L., C.R., C.N., K.O., M.W., S.K., M.M., R.V., M.P., M.-H.J., O.-J.K., R.V., and C.N.K. designed and performed the experiments. Y.O., Z.L., O.-J.K., and C.N.K. wrote the manuscript, with help from all authors.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Yuriko Osakabe or Chidananda Nagamangala Kanchiswamy.

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DOI

https://doi.org/10.1038/s41596-018-0067-9

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