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Microinjection pp 179-190 | Cite as

Delivery of CRISPR-Cas9 into Mouse Zygotes by Electroporation

  • Wenning Qin
  • Haoyi Wang
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1874)

Abstract

The CRISPR-Cas9 system in bacteria and archaea has recently been exploited for genome editing in various model organisms, including the mice. In this scheme, components of the CRISPR-Cas9 system are delivered into the mouse zygote and mutant mice carrying genetic modifications derived. Although microinjection has been the technology of choice, electroporation has also emerged and been proven to be effective delivering CRISPR-Cas9 reagents into the mouse zygote. Here, we describe the experimental protocol employing electroporation to deliver CRISPR-Cas9 reagents into mouse embryos and derive gene-edited mutant mice.

Key words

CRISPR-Cas9 Electroporation Genome editing Mouse models 

Notes

Acknowledgment

We would like to thank the Genetic Engineering Technologies and the Reproductive sciences groups of the Jackson Laboratory for their partnership and contribution exploring into the CRISPR-Cas9 technology. Research reported in this publication was partially supported by the National Cancer Institute under award number P30CA034196. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. H.W. is supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDA16010205), “National Natural Science Foundation of China” (31471215), and the National Key Research and Development Program of China (No. 2016YFA0101402).

References

  1. 1.
    Horvath P, Barrangou R (2010) CRISPR/Cas, the immune system of bacteria and archaea. Science 327(5962):167–170CrossRefGoogle Scholar
  2. 2.
    Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823 http://www.ncbi.nlm.nih.gov/pubmed/23287718%5Cnhttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=PMC3795411CrossRefGoogle Scholar
  3. 3.
    Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA – guided. Science 337:816–822Google Scholar
  4. 4.
    Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE et al (2013) RNA-guided human genome engineering via Cas9. Science 339(6121):823–826CrossRefGoogle Scholar
  5. 5.
    Hai T, Teng F, Guo R, Li W, Zhou Q (2014) One-step generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Res 24(3):372CrossRefGoogle Scholar
  6. 6.
    Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD et al (2013) Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 31(3):227CrossRefGoogle Scholar
  7. 7.
    Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L et al (2014) Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 156(4):836–843CrossRefGoogle Scholar
  8. 8.
    Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F et al (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/cas-mediated genome engineering. Cell 153(4):910–918CrossRefGoogle Scholar
  9. 9.
    Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R (2013) One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154(6):1370–1379CrossRefGoogle Scholar
  10. 10.
    Qin W, Kutny PM, Maser RS, Dion SL, Lamont JD, Zhang Y et al (2016) Generating mouse models using CRISPR-Cas9-mediated genome editing. Curr Protoc Mouse Biol 6:39–66CrossRefGoogle Scholar
  11. 11.
    Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle FH (1980) Genetic transformation of mouse embryos by microinjection of purified DNA. Proc Natl Acad Sci 77(12):7380–7384CrossRefGoogle Scholar
  12. 12.
    Palmiter RD, Brinster RL, Hammer RE, Trumbauer ME, Rosenfeld MG, Birnberg NC et al (1982) Dramatic growth of mice that develop from eggs microinjected with metallothionein–growth hormone fusion genes. Nature 300(5893):611CrossRefGoogle Scholar
  13. 13.
    Chen S, Lee B, Lee AY-F, Modzelewski AJ, He L (2016) Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes. J Biol Chem 291(28):14457–14467CrossRefGoogle Scholar
  14. 14.
    Hashimoto M, Takemoto T (2015) Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing. Sci Rep 5:11315CrossRefGoogle Scholar
  15. 15.
    Kaneko T, Mashimo T (2015) Simple genome editing of rodent intact embryos by electroporation. PLoS One 10(11):e0142755CrossRefGoogle Scholar
  16. 16.
    Qin W, Dion SL, Kutny PM, Zhang Y, Cheng AW, Jillette NL et al (2015) Efficient CRISPR/Cas9-mediated genome editing in mice by zygote electroporation of nuclease. Genetics 200(2):423–430CrossRefGoogle Scholar
  17. 17.
    Wang W, Kutny PM, Byers SL, Longstaff CJ, DaCosta MJ, Pang C et al (2016) Delivery of Cas9 protein into mouse zygotes through a series of electroporation dramatically increases the efficiency of model creation. J Genet Genomics 43(5):319–327CrossRefGoogle Scholar
  18. 18.
    Wassarman PM (1988) Zona pellucida glycoproteins. Annu Rev Biochem 57(1):415–442CrossRefGoogle Scholar
  19. 19.
    Legge M (1995) Oocyte and zygote zona pellucida permeability to macromolecules. J Exp Zool Part A Ecol Genet Physiol 271(2):145–150CrossRefGoogle Scholar
  20. 20.
    Nagy A, Gertsenstein M, Vintersten K, Behringer R (2006) Removal of zona pellucida. Cold Spring Harb Protoc 2006(3):pdb-prot4421Google Scholar
  21. 21.
    Nicolson GL, Yanagimachi R, Yanagimachi H (1975) Ultrastructural localization of lectin-binding sites on the zonae pellucidae and plasma membranes of mammalian eggs. J Cell Biol 66(2):263–274CrossRefGoogle Scholar
  22. 22.
    Escoffre J-M, Portet T, Wasungu L, Teissié J, Dean D, Rols M-P (2009) What is (still not) known of the mechanism by which electroporation mediates gene transfer and expression in cells and tissues. Mol Biotechnol 41(3):286–295CrossRefGoogle Scholar
  23. 23.
    Paganin-Gioanni A, Bellard E, Escoffre JM, Rols MP, Teissie J, Golzio M (2011) Direct visualization at the single-cell level of siRNA electrotransfer into cancer cells. Proc Natl Acad Sci 108(26):10443–10447CrossRefGoogle Scholar
  24. 24.
    Golzio M, Teissié J, Rols M-P (2002) Direct visualization at the single-cell level of electrically mediated gene delivery. Proc Natl Acad Sci 99(3):1292–1297CrossRefGoogle Scholar
  25. 25.
    Byers SL, Payson SJ, Taft RA (2006) Performance of ten inbred mouse strains following assisted reproductive technologies (ARTs). Theriogenology 65(9):1716–1726CrossRefGoogle Scholar
  26. 26.
    Hashimoto M, Yamashita Y, Takemoto T (2016) Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse. Dev Biol 418(1):1–9CrossRefGoogle Scholar
  27. 27.
    Grabarek JB, Plusa B, Glover DM, Zernicka-Goetz M (2002) Efficient delivery of dsRNA into zona-enclosed mouse oocytes and preimplantation embryos by electroporation. Genesis 32(4):269–276CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Wenning Qin
    • 1
  • Haoyi Wang
    • 2
    • 3
  1. 1.The Jackson LaboratoryBar HarborUSA
  2. 2.State Key Laboratory of Stem Cell and Reproductive Biology, Institute of ZoologyChinese Academy of SciencesBeijingChina
  3. 3.Institute for Stem Cell and RegenerationChinese Academy of SciencesBeijingChina

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