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Biolistic Delivery of CRISPR/Cas9 with Ribonucleoprotein Complex in Wheat

  • Zhen Liang
  • Kunling Chen
  • Caixia GaoEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1917)

Abstract

The great advances in exploiting the CRISPR/Cas9 system are paving the way for targeted genome engineering in plants. Genome editing by direct delivery of CRISPR/Cas9 ribonucleoprotein complexes (RNPs) into plant cells reduces off-target mutations and avoids the integration of foreign DNA fragments, thus providing an efficient and accurate method for precision crop breeding. Here we describe an RNP-based genome editing protocol for wheat. The protocol covers the in vitro transcription of sgRNA, purification of Cas9 protein, biolistic delivery of CRISPR/Cas9 RNPs, and tissue culture procedures for regenerating testable seedlings.

Key words

CRISPR/Cas9 Ribonucleoprotein Biolistic delivery Wheat 

Notes

Acknowledgements

This work was supported by grants from the National Key Research and Development Program of China (2016YFD0101804), the National Transgenic Science and Technology Program (2016ZX08010-002), the Chinese Academy of Sciences (QYZDY-SSW-SMC030 and GJHZ1602), and the National Natural Science Foundation of China (31788103, 31420103912 and 31570369).

References

  1. 1.
    Yin K, Gao C, Qiu JL (2017) Progress and prospects in plant genome editing. Nat Plants 3:17107CrossRefGoogle Scholar
  2. 2.
    Belhaj K, Chaparro-Garcia A, Kamoun S, Patron NJ, Nekrasov V (2015) Editing plant genomes with CRISPR/Cas9. Curr Opin Biotechnol 32:76–84CrossRefGoogle Scholar
  3. 3.
    Kouranova E, Forbes K, Zhao G, Warren J, Bartels A, Wu Y, Cui X (2016) CRISPRs for optimal targeting: delivery of CRISPR components as DNA, RNA, and protein into cultured cells and single-cell embryos. Hum Gene Ther 27:464–475CrossRefGoogle Scholar
  4. 4.
    Shan Q, Wang Y, Li J, Gao C (2014) Genome editing in rice and wheat using the CRISPR/Cas system. Nat Protoc 9:2395–2410CrossRefGoogle Scholar
  5. 5.
    Woo JW, Kim J, Kwon SI, Corvalan C, Cho SW, Kim H, Kim SG, Kim ST, Choe S, Kim JS (2015) DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat Biotechnol 33:1162–1164CrossRefGoogle Scholar
  6. 6.
    Zhang Y, Liang Z, Zong Y, Wang Y, Liu J, Chen K, Qiu JL, Gao C (2016) Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nat Commun 7:12617CrossRefGoogle Scholar
  7. 7.
    Staahl BT, Benekareddy M, Coulon-Bainier C, Banfal AA, Floor SN, Sabo JK, Urnes C, Munares GA, Ghosh A, Doudna JA (2017) Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes. Nat Biotechnol 35:431–434CrossRefGoogle Scholar
  8. 8.
    Cho SW, Lee J, Carroll D, Kim JS, Lee J (2013) Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins. Genetics 195:1177–1180CrossRefGoogle Scholar
  9. 9.
    Baek K, Kim DH, Jeong J, Sim SJ, Melis A, Kim JS, Jin E, Bae S (2016) DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins. Sci Rep 6:30620CrossRefGoogle Scholar
  10. 10.
    Hultquist JF, Schumann K, Woo JM, Manganaro L, McGregor MJ, Doudna J, Simon V, Krogan NJ, Marson A (2016) A Cas9 ribonucleoprotein platform for functional genetic studies of HIV-host interactions in primary human T cells. Cell Rep 17:1438–1452CrossRefGoogle Scholar
  11. 11.
    Wu W, Lu Z, Li F, Wang W, Qian N, Duan J, Zhang Y, Wang F, Chen T (2017) Efficient in vivo gene editing using ribonucleoproteins in skin stem cells of recessive dystrophic epidermolysis bullosa mouse model. Proc Natl Acad Sci U S A 114:1660–1665CrossRefGoogle Scholar
  12. 12.
    Subburaj S, Chung SJ, Lee C, Ryu SM, Kim DH, Kim JS, Bae S, Lee GJ (2016) Site-directed mutagenesis in Petunia x hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins. Plant Cell Rep 35:1535–1544CrossRefGoogle Scholar
  13. 13.
    Malnoy M, Viola R, Jung MH, Koo OJ, Kim S, Kim JS, Velasco R, Nagamangala Kanchiswamy C (2016) DNA-free genetically edited grapevine and apple protoplast using crispr/cas9 ribonucleoproteins. Front Plant Sci 7:1904CrossRefGoogle Scholar
  14. 14.
    Kim H, Kim ST, Ryu J, Kang BC, Kim JS, Kim SG (2017) CRISPR/Cpf1-mediated DNA-free plant genome editing. Nat Commun 8:14406CrossRefGoogle Scholar
  15. 15.
    Liang Z, Chen K, Li T, Zhang Y, Wang Y, Zhao Q, Liu J, Zhang H, Liu C, Ran Y, Gao C (2017) Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nat Commun 8:14261CrossRefGoogle Scholar
  16. 16.
    Svitashev S, Schwartz C, Lenderts B, Young JK, Mark Cigan A (2016) Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes. Nat Commun 7:13274CrossRefGoogle Scholar
  17. 17.
    Wolter F, Puchta H (2017) Knocking out consumer concerns and regulator’s rules: efficient use of CRISPR/Cas ribonucleoprotein complexes for genome editing in cereals. Genome Biol 18:43CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

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

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