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CRISPR/Cas9-Mediated Multiply Targeted Mutagenesis in Orange and Purple Carrot Plants

  • Zhi-Sheng Xu
  • Kai Feng
  • Ai-Sheng Xiong
Original Paper
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Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system has been successfully used for precise genome editing in many plant species, including in carrot cells, very recently. However, no stable gene-editing carrot plants were obtained with CRISPR/Cas9 system to date. In the present study, four sgRNA expression cassettes, individually driven by four different promoters and assembled in a single CRISPR/Cas9 vector, were transformed into carrots using Agrobacterium-mediated genetic transformation. Four sites of DcPDS and DcMYB113-like genes were chosen as targets. Knockout of DcPDS in orange carrot ‘Kurodagosun’ resulted in the generation of albino carrot plantlets, with about 35.3% editing efficiency. DcMYB113-like was also successfully edited in purple carrot ‘Deep purple’, resulting in purple depigmented carrot plants, with about 36.4% rate of mutation. Sequencing analyses showed that insertion, deletion, and substitution occurred in the target sites, generating heterozygous, biallelic, and chimeric mutations. The highest efficiency of mutagenesis was observed in the sites targeted by AtU6-29-driven sgRNAs in both DcPDS- and DcMYB113-like-knockout T0 plants, which always induced double-strand breaks in the target sites. Our results proved that CRISPR/Cas9 system could be for generating stable gene-editing carrot plants.

Keywords

CRISPR/Cas9 Carrot Genome editing PDS gene MYB gene Promoter-driven sgRNA 

Notes

Acknowledgements

The authors thank Prof. Yao-Guang Liu (South China Agriculture University, Guangzhou, China) for providing the plant binary vector pYLCRISPR/Cas9 and the sgRNA plasmids. The research was supported by the National Natural Science Foundation of China (Grant Nos. 31501775; 31872098), Open Project of State Key Laboratory of Crop Genetics and Germplasm Enhancement (Grant No. ZW201710) and Priority Academic Program Development of Jiangsu Higher Education Institutions (Grant No. PAPD).

Compliance with Ethical Standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

12033_2018_150_MOESM1_ESM.doc (34 kb)
Supplementary material 1 (DOC 34 KB)

References

  1. 1.
    Vogel, G. (2005). How does a single somatic cell become a whole plant? Science, 309, 86.Google Scholar
  2. 2.
    Kammerer, D., Carle, R., & Schieber, A. (2004). Quantification of anthocyanins in black carrot extracts (Daucus carota ssp. sativus var. atrorubens Alef.) and evaluation of their color properties. European Food Research and Technology, 219, 479–486.Google Scholar
  3. 3.
    Krinsky, N. I., & Johnson, E. J. (2005). Carotenoid actions and their relation to health and disease. Molecular Aspects of Medicine, 26, 459–516.Google Scholar
  4. 4.
    Clotault, J., Peltier, D., Berruyer, R., Thomas, M., Briard, M., & Geoffriau, E. (2008). Expression of carotenoid biosynthesis genes during carrot root development. Journal of Experimental Botany, 59, 3563–3573.Google Scholar
  5. 5.
    Montilla, E. C., Arzaba, M. R., Hillebrand, S., & Winterhalter, P. (2011). Anthocyanin composition of black carrot (Daucus carota ssp. sativus var. atrorubens Alef.) cultivars Antonina, Beta Sweet, Deep Purple, and Purple Haze. J. Agric. Food. Chem., 59, 3385–3390.Google Scholar
  6. 6.
    Xu, Z. S., Tan, H. W., Wang, F., Hou, X. L., & Xiong, A. S. (2014) CarrotDB: A genomic and transcriptomic database for carrot. Database (Oxford), 2014, 1229–1245.Google Scholar
  7. 7.
    Iorizzo, M., Ellison, S., Senalik, D., Zeng, P., Satapoomin, P., Huang, J., Bowman, M., Iovene, M., Sanseverino, W., Cavagnaro, P., Yildiz, M., Macko-Podgorni, A., Moranska, E., Grzebelus, E., Grzebelus, D., Ashrafi, H., Zheng, Z., Cheng, S., Spooner, D., Van Deynze, A., & Simon, P. (2016). A high-quality carrot genome assembly provides new insights into carotenoid accumulation and asterid genome evolution. Nature Genetics, 48, 657–666.Google Scholar
  8. 8.
    Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S., & Gregory, P. D. (2010). Genome editing with engineered zinc finger nucleases. Nature Reviews Genetics, 11, 636–646.Google Scholar
  9. 9.
    Joung, J. K., & Sander, J. D. (2013). TALENs: A widely applicable technology for targeted genome editing. Nature Reviews Molecular Cell Biology., 14, 49–55.Google Scholar
  10. 10.
    Pennisi, E. (2013). The CRISPR craze. Science, 341, 833–836.Google Scholar
  11. 11.
    Johnson, R. D., & Jasin, M. (2001). Double-strand-break-induced homologous recombination in mammalian cells. Biochemical Society Transactions, 29, 196–201.Google Scholar
  12. 12.
    Sonoda, E., Hochegger, H., Saberi, A., Taniguchi, Y., & Takeda, S. (2006). Differential usage of non-homologous end-joining and homologous recombination in double strand break repair. DNA Repair, 5, 1021–1029.Google Scholar
  13. 13.
    Feng, Z. Y., Zhang, B. T., Ding, W. N., Liu, X. D., Yang, D. L., Wei, P. L., Cao, F. Q., Zhu, S. H., Zhang, F., Mao, Y. F., & Zhu, J. K. (2013). Efficient genome editing in plants using a CRISPR/Cas system. Cell Research, 23, 1229–1232.Google Scholar
  14. 14.
    Li, J. F., Norville, J. E., Aach, J., McCormack, M., Zhang, D. D., Bush, J., Church, G. M., & Sheen, J. (2013). Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nature Biotechnology, 31, 688–691.Google Scholar
  15. 15.
    Nekrasov, V., Staskawicz, B., Weigel, D., Jones, J. D. G., & Kamoun, S. (2013). Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nature Biotechnology, 31, 691–693.Google Scholar
  16. 16.
    Miao, J., Guo, D. S., Zhang, J. Z., Huang, Q. P., Qin, G. J., Zhang, X., Wan, J. M., Gu, H. Y., & Qu, L. J. (2013). Targeted mutagenesis in rice using CRISPR-Cas system. Cell Research, 23, 1233–1236.Google Scholar
  17. 17.
    Tang, X., Lowder, L. G., Zhang, T., Malzahn, A. A., Zheng, X., Voytas, D. F., Zhong, Z., Chen, Y., Ren, Q., Li, Q., Kirkland, E. R., Zhang, Y., & Qi, Y. (2017). A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants. Nature Plants, 3, 17108.Google Scholar
  18. 18.
    Wang, M., Mao, Y., Lu, Y., Tao, X., & Zhu, J. K. (2017). Multiplex gene editing in rice using the CRISPR-Cpf1 System. Molecular Plant, 10, 1011–1013.Google Scholar
  19. 19.
    Shan, Q., Wang, Y., Li, J., Zhang, Y., Chen, K., Liang, Z., Zhang, K., Liu, J., Xi, J. J., Qiu, J. L., & Gao, C. (2013). Targeted genome modification of crop plants using a CRISPR-Cas system. Nature Biotechnology, 31, 686–688.Google Scholar
  20. 20.
    Wang, Y. P., Cheng, X., Shan, Q. W., Zhang, Y., Liu, J. X., Gao, C. X., & Qiu, J. L. (2014). Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nature Biotechnology, 32, 947–951.Google Scholar
  21. 21.
    Liang, Z., Zhang, K., Chen, K. L., & Gao, C. X. (2014). Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. Journal of Genetics and Genomics, 41, 63–68.Google Scholar
  22. 22.
    Char, S. N., Neelakandan, A. K., Nahampun, H., Frame, B., Main, M., Spalding, M. H., Becraft, P. W., Meyers, B. C., Walbot, V., Wang, K., & Yang, B. (2017). An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize. Plant Biotechnology Journal, 15, 257–268.Google Scholar
  23. 23.
    Jacobs, T. B., LaFayette, P. R., Schmitz, R. J., & Parrott, W. A. (2015). Targeted genome modifications in soybean with CRISPR/Cas9. BMC Biotechnology, 15, 1–10.Google Scholar
  24. 24.
    Li, Z. S., Liu, Z. B., Xing, A. Q., Moon, B. P., Koellhoffer, J. P., Huang, L. X., Ward, R. T., Clifton, E., Falco, S. C., & Cigan, A. M. (2015). Cas9-guide RNA directed genome editing in soybean. Plant Physiology, 169, 960–970.Google Scholar
  25. 25.
    Wang, S. H., Zhang, S. B., Wang, W. X., Xiong, X. Y., Meng, F. R., & Cui, X. (2015). Efficient targeted mutagenesis in potato by the CRISPR/Cas9 system. Plant Cell Reports, 34, 1473–1476.Google Scholar
  26. 26.
    Jiang, W. Z., Zhou, H. B., Bi, H. H., Fromm, M., Yang, B., & Weeks, D. P. (2013). Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Research, 41, e188.Google Scholar
  27. 27.
    Li, C., Unver, T., & Zhang, B. H. (2017). A high-efficiency CRISPR/Cas9 system for targeted mutagenesis in Cotton (Gossypium hirsutum L.). Scientific Reports, 7, 43902.Google Scholar
  28. 28.
    Brooks, C., Nekrasov, V., Lippman, Z. B., & Van Eck, J. (2014). Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system. Plant Physiology, 166, 1292–1297.Google Scholar
  29. 29.
    Pan, C. T., Ye, L., Qin, L., Liu, X., He, Y. J., Wang, J., Chen, L. F., & Lu, G. (2016). CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations. Scientific Reports, 6, 46916.Google Scholar
  30. 30.
    Jia, H. G., & Wang, N. (2014). Targeted genome editing of sweet orange using Cas9/sgRNA. PLoS ONE, 9, e93806.Google Scholar
  31. 31.
    Nishitani, C., Hirai, N., Komori, S., Wada, M., Okada, K., Osakabe, K., Yamamoto, T., & Osakabe, Y. (2016). Efficient genome editing in apple using a CRISPR/Cas9 system. Scientific Reports, 6, 31481.Google Scholar
  32. 32.
    Nakajima, I., Ban, Y., Azuma, A., Onoue, N., Moriguchi, T., Yamamoto, T., Toki, S., & Endo, M. (2017). CRISPR/Cas9-mediated targeted mutagenesis in grape. PLoS ONE, 12, e0177966.Google Scholar
  33. 33.
    Fan, D., Liu, T. T., Li, C. F., Jiao, B., Li, S., Hou, Y. S., & Luo, K. M. (2015). Efficient CRISPR/Cas9-mediated targeted mutagenesis in populus in the first generation. Scientific Reports, 5, 12217.Google Scholar
  34. 34.
    Oleszkiewicz, M. Klimek-Chodacka,T., Lowder, L. G., Qi, Y., & Baranski, R. (2018). Efficient CRISPR/Cas9-based genome editing in carrot cells. Plant Cell Reports, 37, 575–586.Google Scholar
  35. 35.
    Qin, G. J., Gu, H. Y., Ma, L. G., Peng, Y. B., Deng, X. W., Chen, Z. L., & Qu, L. J. (2007). Disruption of phytoene desaturase gene results in albino and dwarf phenotypes in Arabidopsis by impairing chlorophyll, carotenoid, and gibberellin biosynthesis. Cell Research, 17, 471–482.Google Scholar
  36. 36.
    Li, S. N., Wang, W. Y., Gao, J. L., Yin, K. Q., Wang, R., Wang, C. C., Petersen, M., Mundy, J., & Qiu, J. L. (2016). MYB75 phosphorylation by MPK4 Is required for light-induced anthocyanin accumulation in Arabidopsis. The Plant Cell, 28, 2866–2883.Google Scholar
  37. 37.
    Ma, X., Zhang, Q., Zhu, Q., Liu, W., Chen, Y., Qiu, R., Wang, B., Yang, Z., Li, H., Lin, Y., Xie, Y., Shen, R., Chen, S., Wang, Z., Guo, J., Chen, L., Zhao, X., Dong, Z., & Liu, Y. G. (2015). A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Molecular Plant, 8, 1274–1284.Google Scholar
  38. 38.
    Wang, F., Wang, G. L., Hou, X. L., Li, M. Y., Xu, Z. S., & Xiong, A. S. (2018). The genome sequence of ‘Kurodagosun’, a major carrot variety in Japan and China, reveals insights into biological research and carrot breeding. Molecular Genetics and Genomics, 293, 861–871.Google Scholar
  39. 39.
    Hardegger, M., & Sturm, A. (1998). Transformation and regeneration of carrot (Daucus carota L.). Molecular Breeding, 4, 119–127.Google Scholar
  40. 40.
    Gamborg, O. L., Miller, R. A., & Ojima, K. (1968). Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Research, 50, 151–158.Google Scholar
  41. 41.
    Chen, W. P., & Punja, Z. K. (2002). Transgenic herbicide- and disease-tolerant carrot (Daucus carota L.) plants obtained through Agrobacterium-mediated transformation. Plant Cell Reports, 20, 929–935.Google Scholar
  42. 42.
    Liu, W., Xie, X., Ma, X., Li, J., Chen, J., & Liu, Y. G. (2015). DSDecode: A web-based tool for decoding of sequencing chromatograms for genotyping of targeted mutations. Molecular Plant, 8, 1431–1433.Google Scholar
  43. 43.
    Kim, H., Kim, S. T., Ryu, J., Choi, M. K., Kweon, J., Kang, B. C., Ahn, H. M., Bae, S., Kim, J., Kim, J. S., & Kim, S. G. (2016). A simple, flexible and high-throughput cloning system for plant genome editing via CRISPR-Cas system. Journal of Integrative Plant Biology, 58, 705–712.Google Scholar
  44. 44.
    Cermak, T., Baltes, N. J., Cegan, R., Zhang, Y., & Voytas, D. F. (2015) High-frequency, precise modification of the tomato genome. Genome Biology, 16, 232Google Scholar
  45. 45.
    Gil-Humanes, J., Wang, Y. P., Liang, Z., Shan, Q. W., Ozuna, C. V., Sanchez-Leon, S., Baltes, N. J., Starker, C., Barro, F., Gao, C., & Voytas, D. F. (2017). High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9. The Plant Journal, 89, 1251–1262.Google Scholar
  46. 46.
    Liang, G., Zhang, H. M., Lou, D. J., & Yu, D. Q. (2016) Selection of highly efficient sgRNAs for CRISPR/Cas9-based plant genome editing. Scientific Reports-UK, 6, 21451.Google Scholar
  47. 47.
    Ye, M., Peng, Z., Tang, D., Yang, Z., Li, D., Xu, Y., Zhang, C., & Huang, S. (2018) Generation of self-compatible diploid potato by knockout of S-RNase. Nature Plants, 4, 651Google Scholar
  48. 48.
    Clasen, B. M., Stoddard, T. J., Luo, S., Demorest, Z. L., Li, J., Cedrone, F., Tibebu, R., Davison, S., Ray, E. E., Daulhac, A., Coffman, A., Yabandith, A., Retterath, A., Haun, W., Baltes, N. J., Mathis, L., Voytas, D. F., & Zhang, F. (2016). Improving cold storage and processing traits in potato through targeted gene knockout. Plant Biotechnology Journal, 14, 169–176.Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of HorticultureNanjing Agricultural UniversityNanjingChina

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