Advertisement

Visual Assay for Gene Editing Using a CRISPR/Cas9 System in Carrot Cells

  • Magdalena Klimek-Chodacka
  • Tomasz Oleszkiewicz
  • Rafal Baranski
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1917)

Abstract

The development of the Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas9) system has advanced genome editing and has become widely adopted for this purpose in many species. Its efficient use requires the method adjustment and optimization. Here, we show the use of a model carrot callus system for demonstrating gene editing via CRISPR/Cas9 targeted mutagenesis. The system relies on the utilization of carrot tissue accumulating anthocyanin pigments responsible for a deep purple cell color and generation of knockout mutations in the flavanone-3-hydroxylase (F3H) gene in the anthocyanin biosynthesis pathway. F3H mutant cells targeted by Cas9/gRNA complexes are not able to synthesize anthocyanins and remain white, easily visually distinguished from purple wild-type cells. Mutations are either small indels or larger chromosomal deletions that can be identified by restriction fragment analysis and sequencing. This feasible system can also be applied for validating efficiency of CRISPR/Cas9 vectors.

Key words

Agrobacterium-mediated transformation Daucus carota Callus Anthocyanins Flavanone-3-hydroxylase F3H 

Notes

Acknowledgments

This work was supported by the National Science Centre, Poland (UMO-2013/09/B/NZ9/02379) and by the Ministry of Science and Higher Education of the Republic of Poland.

References

  1. 1.
    Baranski R (2008) Genetic transformation of carrot (Daucus carota) and other Apiaceae species. Transgenic Plant J 2:18–38Google Scholar
  2. 2.
    Iorizzo M, Ellison S, Senalik D, Zeng P, Satapoomin P, Huang J, Bowman M, Iovene M, Sanseverino W, Cavagnaro P, Yildiz M, Spooner DM, Simon PW et al (2016) A high-quality carrot genome assembly provides new insights into carotenoid accumulation and asterid genome evolution. Nat Genet 48:657–666CrossRefGoogle Scholar
  3. 3.
    Klimek-Chodacka M, Oleszkiewicz T, Lowder LG, Qi Y, Baranski R (2018) Efficient CRISPR/Cas9 based genome editing in carrot cells. Plant Cell Rep 37:575–558CrossRefGoogle Scholar
  4. 4.
    Andersson M, Turesson H, Nicolia A, Fält AS, Samuelsson M, Hofvander P (2017) Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts. Plant Cell Rep 36:117–128CrossRefGoogle Scholar
  5. 5.
    Meng Y, Hou Y, Wang H, Ji R, Liu B, Wen J, Niu L, Lin H (2017) Targeted mutagenesis by CRISPR/Cas9 system in the model legume Medicago truncatula. Plant Cell Rep 36:371–374CrossRefGoogle Scholar
  6. 6.
    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 337:816–821CrossRefGoogle Scholar
  7. 7.
    Anders C, Niewoehner O, Duerst A, Jinek M (2014) Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513:569–573CrossRefGoogle Scholar
  8. 8.
    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(1):39CrossRefGoogle Scholar
  9. 9.
    Zaidi SS, Tashkandi M, Mansoor S, Mahfouz MM (2016) Engineering plant immunity: using CRISPR/Cas9 to generate virus resistance. Front Plant Sci 7:1673CrossRefGoogle Scholar
  10. 10.
    Petrussa E, Braidot E, Zancani M, Peresson C, Bartolini A, Patui S, Vianello A (2013) Plant flavonoids—biosynthesis, transport and involvement in stress responses. Int J Mol Sci 14:14950–14973CrossRefGoogle Scholar
  11. 11.
    Cheynier V, Comte G, Davies KM, Lattanzio V, Martens S (2013) Plant phenolics: recent advances on their biosynthesis, genetics, and ecophysiology. Plant Physiol Biochem 72:1–20CrossRefGoogle Scholar
  12. 12.
    Zuker A, Tzfira T, Ben-Meir H, Ovadis M, Shklarman E, Itzhaki H et al (2002) Modification of flower color and fragrance by antisense suppression of the flavanone 3-hydroxylase gene. Mol Breed 9:33–41CrossRefGoogle Scholar
  13. 13.
    Gamborg OL, Miller RA, Ojima A (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158CrossRefGoogle Scholar
  14. 14.
    Lowder LG, Zhang D, Baltes NJ, Paul JW, Tang X, Zheng X, Voytas DF, Hsieh TF, Zhang Y, Qi Y (2015) A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiol 169:971–985CrossRefGoogle Scholar
  15. 15.
    Rogers SO, Bendich AJ (1988) Extraction of DNA from plant tissues. In: Plant molecular biology manual, vol. A6, p. 1–10.CrossRefGoogle Scholar
  16. 16.
    Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133:462–469CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Magdalena Klimek-Chodacka
    • 1
  • Tomasz Oleszkiewicz
    • 1
  • Rafal Baranski
    • 1
  1. 1.Faculty of Biotechnology and Horticulture, Institute of Plant Biology and BiotechnologyUniversity of Agriculture in KrakowKrakowPoland

Personalised recommendations