Advertisement

Application of CRISPR/Cas9-Mediated Gene Editing in Tomato

  • Nathan T. Reem
  • Joyce Van EckEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1917)

Abstract

CRISPR-/Cas9-mediated gene editing has been demonstrated in a number of food crops including tomato. Tomato (Solanum lycopersicum) is both an important food crop and a model plant species that has been used extensively for studying gene function, especially as it relates to fruit biology. This duality in purpose combined with readily available resources (mutant populations, genome sequences, transformation methodology) makes tomato an ideal candidate for gene editing. The CRISPR/Cas9 system routinely used in our laboratory has been applied to various tomato genotypes and the wild species, Solanum pimpinellifolium. The vector system is based on Golden Gate cloning techniques. Cassettes that contain the neomycin phosphotransferase II (NPTII) selectable marker gene that confers resistance to kanamycin, a human codon-optimized Cas9 driven by the CaMV 35S promoter, and guide RNA (gRNA) under control of the Arabidopsis U6 polymerase promoter are assembled into a T-DNA vector. Generally, we design CRISPR/Cas9 constructs that contain two gRNAs per gene target. However, we have been successful with inclusion of up to eight gRNAs to simultaneously target multiple genes and regions. Introduction of CRISPR-/Cas9-designed constructs into tomato is accomplished by transformation methodology based on Agrobacterium tumefaciens infection of young cotyledon sections and selection on kanamycin-containing medium based on the presence of the NPTII gene. The approaches for the development of CRISPR/Cas9 constructs and genotypic analyses (PCR-based amplicon sequencing and T7 endonuclease) are detailed in this chapter.

Key words

Genome editing CRISPR/Cas9 PCR amplicon sequencing Solanaceae Solanum lycopersicum Solanum pimpinellifolium T7 endonuclease 1 (T7E1) assay 

Notes

Acknowledgments

The authors acknowledge the National Science Foundation Plant Genome Research Program (IOS-1732253) for support related to their research on gene editing in tomato. The authors would like to thank Sophien Kamoun and Vlad Nekrasov for the guidance during the early development of gene editing methodology for tomato with their vector system. The authors also acknowledge the members of the Lippman lab at Cold Spring Harbor Laboratory and members of the Van Eck lab affiliated with tomato transformation. Michelle Tjahjadi provided the flowchart included in Fig. 1.

References

  1. 1.
    Doudna JA, Charpentier E (2014) Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346:1–9CrossRefGoogle Scholar
  2. 2.
    Sato S, Tabata S (2016) Tomato genome sequence. In: Ezura H, Ariizumi T, Garcia-Mas J, Rose J (eds) Functional genomics and biotechnology in Solanaceae and Cucurbitaceae crops. Springer Berlin Heidelberg, BerlinGoogle Scholar
  3. 3.
    Kumar R, Khurana A (2014) Functional genomics of tomato: opportunities and challenges in post-genome NGS era. J Biosci 39:917–929CrossRefGoogle Scholar
  4. 4.
    Van Eck J, Tjahjadi M, Keen K (2018) Agrobacterium tumefaciens-mediated transformation of tomato. In: Kumar S, Barone P, Smith M (eds) Transgenic plants: methods and protocols. Springer Science+Business Media, LLC, New YorkGoogle Scholar
  5. 5.
  6. 6.
    Van Eck J (2017) Gene editing in tomatoes. Emerg Top Life Sci 1:183–191CrossRefGoogle Scholar
  7. 7.
    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 166:1292–1297CrossRefGoogle Scholar
  8. 8.
    Ron M, Kajala K, Pauluzzi G, Wang D, Reynoso MA, Zumstein K, Garcha J, Winte S, Masson H, Inagaki S, Federici F, Sinha N, Deal RB, Bailey-Serres J, Brady SM (2014) Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model. Plant Physiol 166:455–469CrossRefGoogle Scholar
  9. 9.
    Pan C, Ye L, Qin L, Liu X, He Y, Wang J, Chen L, Lu G (2016) CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations. Sci Rep 6:1–9CrossRefGoogle Scholar
  10. 10.
    Belhaj K, Chaparro-Garcia A, Kamoun S, Nekrasov V (2013) Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods 9:39–48CrossRefGoogle Scholar
  11. 11.
    Nekrasov V, Wang C, Win J, Lanz C, Weigel D, Kamoun S (2017) Rapid generation of a transgene-free powdery mildew resistant tomato by genome deletion. Sci Rep 7:482–487CrossRefGoogle Scholar
  12. 12.
    Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, Wang L, Hodgkins A, Iyer V, Huang X, Skarnes WC (2014) Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat Methods 11:399–402CrossRefGoogle Scholar
  13. 13.
    Lei Y, Lu L, Liu HY, Li S, Xing F, Chen LL (2014) CRISPR-P: a web tool for synthetic single-guide RNA design of CRISPR-system in plants. Mol Plant 7:1494–1496CrossRefGoogle Scholar
  14. 14.
    Naito Y, Hino K, Bono H, Ui-Tei K (2015) CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics 31:1120–1123CrossRefGoogle Scholar
  15. 15.
    Clarke JD (2009) Cetyltrimethyl ammonium bromide (CTAB) DNA miniprep for plant DNA isolation. Cold Spring Harb Protoc 4:1–3Google Scholar
  16. 16.
    Auer TO, Duroure K, Concordet JP, Del Bene F (2014) CRISPR/Cas9-mediated conversion of eGFP- into Gal4-transgenic lines in zebrafish. Nat Protoc 9:2823–2840CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Boyce Thompson InstituteIthacaUSA
  2. 2.Plant Breeding and Genetics Section, School of Integrative Plant ScienceCornell UniversityIthacaUSA

Personalised recommendations