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CRISPR–Cas9-mediated genome editing in apple and grapevine

Nature Protocols volume 13, pages 2844–2863 (2018)Cite this article

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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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Fig. 1: Schematic diagram of plasmid-mediated CRISPR–Cas9-based gene editing, and gene editing by direct delivery of CRISPR–Cas9 RNPs to plant cells to produce genetically edited crop plants.
Fig. 2: Anticipated results of plasmid-mediated CRISPR–Cas9-based gene editing in apple.
Fig. 3: Anticipated results of plasmid-mediated CRISPR–Cas9-based gene editing in grape.
Fig. 4: Anticipated results for CRISPR–Cas9-based editing in apple and grapevine by RNP delivery.

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References

  1. Osakabe, Y., Osakabe, K., Shinozaki, K. & Tran, L. S. P. Response of plants to water stress. Front. Plant Sci. 5, 86 (2014).

    Article  Google Scholar 

  2. Kanchiswamy, C. N. et al. Looking forward to genetically edited fruit crops. Trends Biotechnol. 33, 62–64 (2015).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Song, G. et al. CRISPR/Cas9: a powerful tool for crop genome editing. Crop J. 4, 75–82 (2016).

    Article  Google Scholar 

  5. Zhang, Y. et al. Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nat. Commun. 7, 12617 (2016).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  7. Jia, H. & Wang, N. Targeted genome editing of sweet orange using Cas9/sgRNA. PLoS ONE 9, e93806 (2014).

    Article  Google Scholar 

  8. Fan, D. et al. Efficient CRISPR–Cas9-mediated targeted mutagenesis in Populus in the first generation. Sci. Rep. 5, 12217 (2015).

    Article  CAS  Google Scholar 

  9. Nishitani, C. et al. Efficient genome editing in apple using a CRISPR–Cas9 system. Sci. Rep. 6, 31481 (2016).

    Article  CAS  Google Scholar 

  10. Ren, C. et al. CRISPR–Cas9-mediated efficient targeted mutagenesis in Chardonnay (Vitis vinifera L.). Sci. Rep. 6, 32289 (2016).

    Article  CAS  Google Scholar 

  11. 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 

  12. GóMez-Lim, M. A. & Litz, R. E. Genetic transformation of perennial tropical fruits. In Vitro Cell. Dev. Biol. Plant 40, 442–449 (2004).

    Article  Google Scholar 

  13. Gambino, G. & Gribaudo, I. Genetic transformation of fruit trees: current status and remaining challenges. Transgenic Res. 21, 1163–1181 (2012).

    Article  CAS  Google Scholar 

  14. Kim, H. CRISPR/Cpf1-mediated DNA-free plant genome editing. Nat. Commun. 8, 14406 (2017).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. Velasco, R. et al. The genome of the domesticated apple (Malus x domestica Borkh.). Nat. Genet. 42, 833–839 (2010).

    Article  CAS  Google Scholar 

  18. Jaillon, O. et al. The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature 449, 463–467 (2007).

    Article  CAS  Google Scholar 

  19. Patat-Ochatt, E. M., Boccon-Gibod, J., Duron, M. & Ochatt, S. J. Organogenesis of stem and leaf protoplasts of a haploid golden delicious apple clone (Malus X domestica Borkh.). Plant Cell Rep. 12, 118–120 (1993).

    Article  CAS  Google Scholar 

  20. Doughty, S. & Power, J. B. Callus formation from leaf mesophyll protoplasts of Malus X domestica Borkh. cv. Greensleeves. Plant Cell Rep. 7, 200–201 (1988).

    Article  CAS  Google Scholar 

  21. Lin, Y. C. et al. A simple improved-throughput xylem protoplast system for studying wood formation. Nat. Protoc. 9, 2194–2205 (2014).

    Article  CAS  Google Scholar 

  22. Patat-Ochatt, E. M. et al. Organogenesis of stem and leaf protoplasts of a haploid golden delicious apple clone (Malus X domestica Borkh.). Plant Cell Rep. 12, 118–120 (1993).

    Article  CAS  Google Scholar 

  23. Saito, A. & Suzuki, M. Plant regeneration from meristem-derived callus protoplasts of apple (Malus3domestica cv. ‘Fuji’). Plant Cell Rep. 18, 549–553 (1999).

    Article  CAS  Google Scholar 

  24. Zhu, Y.-M., Hoshino, Y., Nakano, M., Takahashi, E. & Mii, M. Highly efficient system of plant regeneration from protoplasts of grapevine (Vitis vinifera L.) through somatic embryogenesis by using embryogenic callus culture and activated charcoal. Plant Sci. 123, 151–157 (1997).

    Article  CAS  Google Scholar 

  25. Tomiczak, K., Mikuła, A., Sliwinska, E. & Rybczyński, J. J. Autotetraploid plant regeneration by indirect somatic embryogenesis from leaf mesophyll protoplasts of diploid Gentiana decumbens L.f. In Vitro Cell. Dev. Biol. 51, 350–359 (2015).

    Article  CAS  Google Scholar 

  26. Gelvin, B. Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol. Mol. Biol. Rev. 67, 16–37 (2003).

    Article  CAS  Google Scholar 

  27. Wada, M. et al. Anatomical analysis by two approaches ensure the promoter activities of apple AFL genes. J. Jpn. Soc. Hortic. Sci. 78, 32–39 (2009).

    Article  CAS  Google Scholar 

  28. Stamp, J. A. & Meredith, C. P. Somatic embryogenesis from leaves and anthers of grapevine. Sci. Hortic. 35, 235–250 (1988).

    Article  Google Scholar 

  29. Nakano, M., Watanabe, Y. & Hoshino, Y. Histological examination of callogenesis and adventitious embryogenesis in immature ovary culture of grapevine (Vitis vinifera L.). J. Hortic. Sci. Biotechnol. 75, 154–160 (2000).

    Article  Google Scholar 

  30. Dai, L. et al. Establishment of a picloram-induced somatic embryogenesis system in Vitis vinifera cv. chardonnay and genetic transformation of a stilbene synthase gene from wild-growing Vitis species. Plant Cell Tissue Organ Cult. 121, 397–412 (2015).

    Article  CAS  Google Scholar 

  31. Kanchiswamy, C. N., Malnoy, M., Velasco, R., Kim, J. S. & Viola, R. Non-GMO genetically edited crop plants. Trends Biotechnol. 9, 489–491 (2015).

    Article  Google Scholar 

  32. Jelly, N. S., Valat, L., Walter, B. & Maillot, P. Transient expression assays in grapevine: a step towards genetic improvement. Plant Biotechnol. J. 12, 1231–1245 (2014).

    Article  Google Scholar 

  33. Kim, S., Kim, D., Cho, S. W., Kim, J. & Kim, J. S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014).

    Article  CAS  Google Scholar 

  34. Xiao, A. et al. CasOT: a genome-wide Cas9/gRNA off-target searching tool. Bioinformatics 30, 1180–1182 (2014).

    Article  CAS  Google Scholar 

  35. Doench, J. G. et al. Rational design of highly active sgRNAs for CRISPR–Cas9-mediated gene inactivation. Nat. Biotechnol. 32, 1262–1267 (2014).

    Article  CAS  Google Scholar 

  36. Ueta, R. et al. Rapid breeding of parthenocarpic tomato plants using CRISPR–Cas9. Sci. Rep. 7, 507 (2017).

    Article  Google Scholar 

  37. Sawada, H., Ieki, H. & Matsuda, I. PCR detection of Ti and Ri plasmids from phytopathogenic Agrobacterium strains. Appl. Environ. Microbiol. 61, 828–831 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Höfgen, R. & Willmitzer, L. Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res. 16, 9877 (1988).

    Article  Google Scholar 

  39. Edwards, K., Johnstone, C. & Thompson, C. A simple and rapid method for the preparation of plant genomic DNA for PCRanalysis. Nucleic Acid Res. 19, 1349 (1991).

    Article  CAS  Google Scholar 

  40. 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  Google Scholar 

  41. Liu, J. et al. Efficient delivery of nuclease proteins for genome editing in human stem cells and primary cells. Nat. Protoc. 10, 1842–1859 (2015).

    Article  CAS  Google Scholar 

  42. Cho, S. W., Kim, S., Kim, J. M. & Kim, J. S. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 230–232 (2013).

    Article  CAS  Google Scholar 

  43. Lei, R. et al. A simple and effective method to encapsulate tobacco mesophyll protoplasts to maintain cell viability. MethodsX 2, 24–32 (2015).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

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

  1. Riccardo Velasco

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

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

Authors and 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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  1. Yuriko Osakabe
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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.

Corresponding authors

Correspondence to Yuriko Osakabe or Chidananda Nagamangala Kanchiswamy.

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

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Osakabe, Y., Liang, Z., Ren, C. et al. CRISPR–Cas9-mediated genome editing in apple and grapevine. Nat Protoc 13, 2844–2863 (2018). https://doi.org/10.1038/s41596-018-0067-9

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