Advertisement

Targeted Mutagenesis in Hexaploid Bread Wheat Using the TALEN and CRISPR/Cas Systems

  • Yanpeng Wang
  • Yuan Zong
  • Caixia GaoEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1679)

Abstract

The use of sequence-specific transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats-associated system (CRISPR/Cas9) have provided powerful reverse genetic approaches to the targeted modification of genomes in numerous organisms. Both systems have been employed to generate loss-of-function alleles in bread wheat, by targeting multiple and single copies of genes. Here we present protocols for modifying the wheat genome using the two systems. The protocols include the design of TALEN and CRISPR/Cas9 target sites and their construction, evaluation of their activities in protoplasts, transformation of plants, and mutation screening.

Key words

Wheat Genome editing TALEN CRISPR/Cas9 Wheat protoplasts Targeted mutagenesis 

Notes

Acknowledgments

This work was supported by grants from the National Key Research and Development Program of China (2016YFD0101804), the Chinese Academy of Sciences (KFZD-SW-107 and GJHZ1602).

References

  1. 1.
    Dvořák J (2009) Triticeae genome structure and evolution. In: Muehlbauer GJ, Feuillet C (eds) Genetics and genomics of the triticeae. Springer, New York, pp 685–711Google Scholar
  2. 2.
    Sallaud C, Gay C, Larmande P, Bès M, Piffanelli P, Piégu B, Droc G, Regad F, Bourgeois E, Meynard D et al (2004) High throughput T-DNA insertion mutagenesis in rice: a first step towards in silico reverse genetics. Plant J 39:450–464CrossRefPubMedGoogle Scholar
  3. 3.
    Uauy C, Paraiso F, Colasuonno P, Tran RK, Tsai H, Berardi S, Comai L, Dubcovsky J (2009) A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat. BMC Plant Biol 9:115CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Lukens L, Fitzgerald TL, Powell JJ, Stiller J, Weese TL, Abe T, Zhao G, Jia J, McIntyre CL, Li Z et al (2015) An assessment of heavy ion irradiation mutagenesis for reverse genetics in wheat (Triticum aestivum L.) PLoS One 10:e0117369CrossRefGoogle Scholar
  5. 5.
    Bibikova M, Beumer K, Trautman JK, Carroll D (2003) Enhancing Gene targeting with designed zinc finger nucleases. Science 300:764CrossRefPubMedGoogle Scholar
  6. 6.
    Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, Lahaye T, Nickstadt A, Bonas U (2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326:1509–1512CrossRefPubMedGoogle Scholar
  7. 7.
    Moscou MJ, Bogdanove AJ (2009) A simple cipher governs DNA recognition by TAL effectors. Science 326:1501CrossRefPubMedGoogle Scholar
  8. 8.
    Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    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
  10. 10.
    Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Bibikova M, Carroll D, Segal DJ, Trautman JK, Smith J, Kim YG, Chandrasegaran S (2001) Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol Cell Biol 21:289–297CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Chen K, Gao C (2013) Targeted genome modification technologies and their applications in crop improvements. Plant Cell Rep 33:575–578Google Scholar
  13. 13.
    Kim H, Kim JS (2014) A guide to genome engineering with programmable nucleases. Nat Rev Genet 15:321–334CrossRefPubMedGoogle Scholar
  14. 14.
    Bogdanove AJ, Voytas DF (2011) TAL effectors: customizable proteins for DNA targeting. Science 333:1843–1846CrossRefPubMedGoogle Scholar
  15. 15.
    Mak AN, Bradley P, Cernadas RA, Bogdanove AJ, Stoddard BL (2012) The crystal structure of TAL effector PthXo1 bound to its DNA target. Science 335:716–719CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia NV, Bogdanove AJ, Voytas DF (2011) Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res 39:e82CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Sander JD, Cade L, Khayter C, Reyon D, Peterson RT, Joung JK, Yeh J-RJ (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nat Biotech 29:697–698CrossRefGoogle Scholar
  18. 18.
    Reyon D, Tsai SQ, Khayter C, Foden JA, Sander JD, Joung JK (2012) FLASH assembly of TALENs for high-throughput genome editing. Nat Biotech 30:460–465CrossRefGoogle Scholar
  19. 19.
    Zhang Y, Zhang F, Li X, Baller JA, Qi Y, Starker CG, Bogdanove AJ, Voytas DF (2013) Transcription activator-like effector nucleases enable efficient plant genome engineering. Plant Physiol 161:20–27CrossRefPubMedGoogle Scholar
  20. 20.
    Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, Qiu J-L (2014a) Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat Biotech 32:947–951CrossRefGoogle Scholar
  21. 21.
    Shan Q, Wang Y, Li J, Gao C (2014) Genome editing in rice and wheat using the CRISPR/Cas system. Nat Protocols 9:2395–2410CrossRefPubMedGoogle Scholar
  22. 22.
    Zhang K, Liu J, Zhang Y, Yang Z, Gao C (2015) Biolistic genetic transformation of a wide range of Chinese elite wheat (Triticum aestivum L.) varieties. J Genet Genomics 42:39–42CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.State Key Laboratory of Plant Cell and Chromosome Engineering, and Center for Genome Editing, Institute of Genetics and Developmental BiologyChinese Academy of SciencesBeijingChina
  2. 2.University of Chinese Academy of SciencesBeijingChina

Personalised recommendations