Plant Cell Reports

, Volume 36, Issue 3, pp 399–406 | Cite as

Efficient CRISPR/Cas9-based gene knockout in watermelon

  • Shouwei Tian
  • Linjian Jiang
  • Qiang Gao
  • Jie Zhang
  • Mei Zong
  • Haiying Zhang
  • Yi Ren
  • Shaogui Guo
  • Guoyi Gong
  • Fan Liu
  • Yong Xu
Original Article

Abstract

Key message

CRISPR/Cas9 system can precisely edit genomic sequence and effectively create knockout mutations in T0 generation watermelon plants.

Abstract

Genome editing offers great advantage to reveal gene function and generate agronomically important mutations to crops. Recently, RNA-guided genome editing system using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been applied to several plant species, achieving successful targeted mutagenesis. Here, we report the genome of watermelon, an important fruit crop, can also be precisely edited by CRISPR/Cas9 system. ClPDS, phytoene desaturase in watermelon, was selected as the target gene because its mutant bears evident albino phenotype. CRISPR/Cas9 system performed genome editing, such as insertions or deletions at the expected position, in transfected watermelon protoplast cells. More importantly, all transgenic watermelon plants harbored ClPDS mutations and showed clear or mosaic albino phenotype, indicating that CRISPR/Cas9 system has technically 100% of genome editing efficiency in transgenic watermelon lines. Furthermore, there were very likely no off-target mutations, indicated by examining regions that were highly homologous to sgRNA sequences. Our results show that CRISPR/Cas9 system is a powerful tool to effectively create knockout mutations in watermelon.

Keywords

CRISPR/Cas9 Genome editing PDS Watermelon 

Supplementary material

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Supplementary material 1 (PDF 11 kb)
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Supplementary material 2 (PDF 6031 kb)
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Supplementary material 3 (PDF 11016 kb)
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Supplementary material 4 (PDF 102 kb)
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Supplementary material 5 (PDF 114 kb)

References

  1. Brooks C, Nekrasov V, Lippman ZB, 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 Physiol 166:1292–1297CrossRefPubMedPubMedCentralGoogle Scholar
  2. Cardi T, Stewart CN Jr (2016) Progress of targeted genome modification approaches in higher plants. Plant Cell Rep 35:1401–1416CrossRefPubMedGoogle Scholar
  3. Chandrasekaran J, Brumin M, Wolf D, Leibman D, Klap C, Pearlsman M, Sherman A, Arazi T, Gal-On A (2016) Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 technology. Mol Plant Pathol 17:1140–1153CrossRefPubMedGoogle Scholar
  4. Cho MA, Moon CY, Liu JR, Choi PS (2008) Agrobacterium-mediated transformation in Citrullus lanatus. Biol Plant 52:365–369CrossRefGoogle Scholar
  5. Choi PS, Soh WY, Kim YS, Yoo OJ, Liu JR (1994) Genetic transformation and plant regeneration of watermelon using Agrobacterium tumefaciens. Plant Cell Rep 13:344–348CrossRefPubMedGoogle Scholar
  6. Ellul P, Rios G, Atares A, Roig LA, Serrano R, Moreno V (2003) The expression of the Saccharomyces cerevisiae HAL1 gene increases salt tolerance in transgenic watermelon [Citrullus lanatus (Thunb.) Matsun. & Nakai.]. Theor Appl Genet 107:462–469CrossRefPubMedGoogle Scholar
  7. Fan D, Liu T, Li C, Jiao B, Li S, Hou Y, Luo K (2015) Efficient CRISPR/Cas9-mediated targeted mutagenesis in Populus in the first generation. Sci Rep 5:12217CrossRefPubMedPubMedCentralGoogle Scholar
  8. Gao J, Wang G, Ma S, Xie X, Wu X, Zhang X, Wu Y, Zhao P, Xia Q (2015) CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum. Plant Mol Biol 87:99–110CrossRefPubMedGoogle Scholar
  9. Guo S, Zhang J, Sun H, Salse J, Lucas WJ, Zhang H, Zheng Y, Mao L, Ren Y, Wang Z, Min J, Guo X, Murat F, Ham BK, Zhang Z, Gao S, Huang M, Xu Y, Zhong S, Bombarely A, Mueller LA, Zhao H, He H, Zhang Y, Zhang Z, Huang S, Tan T, Pang E, Lin K, Hu Q, Kuang H, Ni P, Wang B, Liu J, Kou Q, Hou W, Zou X, Jiang J, Gong G, Klee K, Schoof H, Huang Y, Hu X, Dong S, Liang D, Wang J, Wu K, Xia Y, Zhao X, Zheng Z, Xing M, Liang X, Huang B, Lv T, Wang J, Yin Y, Yi H, Li R, Wu M, Levi A, Zhang X, Giovannoni JJ, Wang J, Li Y, Fei Z, Xu Y (2013) The draft genome of watermelon (Citrullus lanatus) and resequencing of 20 diverse accessions. Nat Genet 45:51–58CrossRefPubMedGoogle Scholar
  10. Huang YC, Chiang CH, Li CM, Yu TA (2011) Transgenic watermelon lines expressing the nucleocapsid gene of Watermelon silver mottle virus and the role of thiamine in reducing hyperhydricity in regenerated shoots. Plant Cell Tissue Organ Cult 106:21–29CrossRefGoogle Scholar
  11. Jia Y, Ding Y, Shi Y, Zhang X, Gong Z, Yang S (2016) The cbfs triple mutants reveal the essential functions of CBFs in cold acclimation and allow the definition of CBF regulons in Arabidopsis. New Phytol 212:345–353CrossRefPubMedGoogle Scholar
  12. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821CrossRefPubMedGoogle Scholar
  13. Lee J, Chung JH, Kim HM, Kim DW, Kim H (2016) Designed nucleases for targeted genome editing. Plant Biotechnol J 14:448–462CrossRefPubMedGoogle Scholar
  14. Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM, Sheen J (2013) Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol 31:688–691CrossRefPubMedPubMedCentralGoogle Scholar
  15. Li J, Meng X, Zong Y, Chen K, Zhang H, Liu J, Li J, Gao C (2016) Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9. Nat Plants 2:16139CrossRefPubMedGoogle Scholar
  16. Liu L, Gu Q, Ijaz R, Zhang J, Ye Z (2016) Generation of transgenic watermelon resistance to Cucumber mosaic virus facilitated by an effective Agrobacterium -mediated transformation method. Sci Hortic 205:32–38CrossRefGoogle Scholar
  17. 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, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu YG (2015) A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant 8:1274–1284CrossRefPubMedGoogle Scholar
  18. Moreno-Mateos MA, Vejnar CE, Beaudoin JD, Fernandez JP, Mis EK, Khokha MK, Giraldez AJ (2015) CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. Nat Methods 12:982–988CrossRefPubMedPubMedCentralGoogle Scholar
  19. Nishibayashi S, Kaneko H, Hayakawa T (1996) Transformation of cucumber (Cucumis sativus L.) plants using Agrobacterium tumefaciens and regeneration from hypocotyl explants. Plant Cell Rep 15:809–814CrossRefPubMedGoogle Scholar
  20. Qin G, Gu H, Ma L, Peng Y, Deng XW, Chen Z, Qu LJ (2007) Disruption of phytoene desaturase gene results in albino and dwarf phenotypes in Arabidopsis by impairing chlorophyll, carotenoid, and gibberellin biosynthesis. Cell Res 17:471–482CrossRefPubMedGoogle Scholar
  21. Ren C, Liu X, Zhang Z, Wang Y, Duan W, Li S, Liang Z (2016) CRISPR/Cas9-mediated efficient targeted mutagenesis in Chardonnay (Vitis vinifera L.). Sci Rep 6:32289CrossRefPubMedPubMedCentralGoogle Scholar
  22. Schaeffer SM, Nakata PA (2016) The expanding footprint of CRISPR/Cas9 in the plant sciences. Plant Cell Rep 35:1451–1468CrossRefPubMedGoogle Scholar
  23. Symington LS, Gautier J (2011) Double-strand break end resection and repair pathway choice. Annu Rev Genet 45:247–271CrossRefPubMedGoogle Scholar
  24. Tsai SQ, Zheng Z, Nguyen NT, Liebers M, Topkar VV, Thapar V, Wyvekens N, Khayter C, Iafrate AJ, Le LP, Aryee MJ, Joung JK (2015) GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol 33:187–197CrossRefPubMedGoogle Scholar
  25. Wang S, Zhang S, Wang W, Xiong X, Meng F, Cui X (2015) Efficient targeted mutagenesis in potato by the CRISPR/Cas9 system. Plant Cell Rep 34:1473–1476CrossRefPubMedGoogle Scholar
  26. Wechter WP, Levi A, Harris KR, Davis AR, Fei Z, Katzir N, Giovannoni JJ, Salman-Minkov A, Hernandez A, Thimmapuram J, Tadmor Y, Portnoy V, Trebitsh T (2008) Gene expression in developing watermelon fruit. BMC Genom 9:275–288CrossRefGoogle Scholar
  27. Xie K, Zhang J, Yang Y (2014) Genome-wide prediction of highly specific guide RNA spacers for CRISPR-Cas9-mediated genome editing in model plants and major crops. Mol Plant 7:923–926CrossRefPubMedGoogle Scholar
  28. Xie K, Minkenberg B, Yang Y (2015) Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci USA 112:3570–3575CrossRefPubMedPubMedCentralGoogle Scholar
  29. Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ (2014) A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol 14:327CrossRefPubMedPubMedCentralGoogle Scholar
  30. Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572CrossRefPubMedGoogle Scholar
  31. Yu TA, Chiang CH, Wu HW, Li CM, Yang CF, Chen JH, Chen YW, Yeh SD (2011) Generation of transgenic watermelon resistant to Zucchini yellow mosaic virus and Papaya ringspot virus type W. Plant Cell Rep 30:359–371CrossRefPubMedGoogle Scholar
  32. Zhang B, Yang X, Yang C, Li M, Guo Y (2016) Exploiting the CRISPR/Cas9 system for targeted genome mutagenesis in Petunia. Sci Rep 6:20315CrossRefPubMedPubMedCentralGoogle Scholar
  33. Zhou H, Liu B, Weeks DP, Spalding MH, Yang B (2014) Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice. Nucleic Acids Res 42:10903–10914CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Shouwei Tian
    • 1
  • Linjian Jiang
    • 2
  • Qiang Gao
    • 3
  • Jie Zhang
    • 1
  • Mei Zong
    • 1
  • Haiying Zhang
    • 1
  • Yi Ren
    • 1
  • Shaogui Guo
    • 1
  • Guoyi Gong
    • 1
  • Fan Liu
    • 1
  • Yong Xu
    • 1
  1. 1.National Engineering Research Center for VegetablesBeijing Academy of Agriculture and Forestry Sciences, Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (North China), Beijing Key Laboratory of Vegetable Germplasm ImprovementBeijingChina
  2. 2.Department of Plant PathologyChina Agricultural UniversityBeijingChina
  3. 3.Beijing University of AgricultureBeijingChina

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