GONAD: A Novel CRISPR/Cas9 Genome Editing Method that Does Not Require Ex Vivo Handling of Embryos

Channabasavaiah B. Gurumurthy1, Gou Takahashi1, Kenta Wada2, Hiromi Miura3, Masahiro Sato4, Masato Ohtsuka3

1 These authors contributed equally to this work, 2 Department of Bioproduction, Tokyo University of Agriculture, Abashiri, Hokkaido, 3 Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, 4 Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 15.8
DOI:  10.1002/0471142905.hg1508s88
Online Posting Date:  January, 2016
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Transgenic technologies used for creating a desired genomic change in animals involve three critical steps: isolation of fertilized eggs, microinjection of transgenic DNA into them and their subsequent transfer to recipient females. These ex vivo steps have been widely used for over 3 decades and they were also readily adapted for the latest genome editing technologies such as ZFNs, TALENs, and CRISPR/Cas9 systems. We recently developed a method called GONAD (Genome editing via Oviductal Nucleic Acids Delivery) that does not require all the three critical steps of transgenesis and therefore relieves the bottlenecks of widely used animal transgenic technologies. Here we provide protocols for the GONAD system. © 2016 by John Wiley & Sons, Inc.

Keywords: GONAD; genome editing; microinjection; transgenic; CRISPR/Cas9; gene delivery; in vivo electroporation

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Figures
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Basic Protocol 1:

  • Genome editing components: single guide (sg) RNA and Cas9 mRNA stock solutions, both at about 2 to 4 μg/μl concentration.
  • 20% isoflurane (Escain, Mylan; usually stored at 4°C) diluted with propylene glycol (Wako, cat. no. 164‐04996) is warmed to room temperature just before use (approximately 20 ml of 20% isoflurane is needed for five mice)
  • Isoflurane‐soaked absorbent cotton balls
  • Pregnant female mice at 1.5 day of gestation
  • Spray bottle with 70% ethanol
  • 0.5% Trypan blue stock solution (Nacalai tesque, cat. no. 29853‐34)
  • Phosphate‐buffered saline without Ca2+ and Mg2+ (PBS)
  • Bottle/jar
  • 35‐ml disposable syringes
  • 1.5‐ml microcentrifuge tubes
  • Paper towels
  • Dissecting microscope (e.g., Olympus SZ11) with a hot plate (e.g., KM‐1, Kitazato)
  • Forceps (e.g., no. 5, INOX: no. A‐7 and A‐10, NAPOX, Natsume)
  • Aorta‐Klemme (30‐mm in length; no. C‐17, NAPOX, Natsume), two
  • Mouth pipetting device (see recipe)
  • KimWipes
  • BTX T820 electroporator, with tweezer‐type electrodes (made from Nepa Gene; www.nepagene.jp/)
  • Suture wound clips (MikRon 9‐mm autoclip; Becton Dickinson)
  • Scissors (Natsume, cat. no. B‐12H)
NOTE: We suggest using a new box of paper towels and KimWipes each time as they are clean and free of any contaminants such as dust and microbes.NOTE: 60‐mm tissue culture dishes can be used as containers for paper towel pieces and KimWipesNOTE: All surgical instruments should be sterilized by immersing them with 70% ethanol.
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Literature Cited

Literature Cited
  Aida, T., Chiyo, K., Usami, T., Ishikubo, H., Imahashi, R., Wada, Y., Tanaka, K. F., Sakuma, T., Yamamoto, T., and Tanaka, K. 2015. Cloning‐free CRISPR/Cas system facilitates functional cassette knock‐in in mice. Genome Biol. 16. Available at: http://genomebiology.com/2015/16/1/87.
  Behringer, R., Gertsenstein, M., Nagy, K. V., and Nagy, A., eds., 2014. Manipulating the Mouse Embryo: A Laboratory Manual. 4th Edition. Cold Spring Harbor Laboratory Press.
  Harms, D. W., Quadros, R. M., Seruggia, D., Ohtsuka, M., Takahashi, G., Montoliu, L., and Gurumurthy, C. B. 2014. Mouse Genome Editing Using the CRISPR/Cas System. Curr. Protoc. Hum. Genet. 83:15.7.1‐15.7.27. doi: 10.1002/0471142905.hg1507s83.
  Horii, T., Arai, Y., Yamazaki, M., Morita, S., Kimura, M., Itoh, M., Abe, Y., and Hatada, I. 2014. Validation of microinjection methods for generating knockout mice by CRISPR/Cas‐mediated genome engineering. Sci. Rep. 4. Available at: http://www.nature.com/doifinder/10.1038/srep04513. doi: 10.1038/srep04513.
  Kaneko, T., Sakuma, T., Yamamoto, T., and Mashimo, T. 2014. Simple knockout by electroporation of engineered endonucleases into intact rat embryos. Sci. Rep. 4:6382. doi: 10.1038/srep06382.
  Liu, C., Xie, W., Gui, C., and Du, Y. 2013. Pronuclear Microinjection and Oviduct Transfer Procedures for Transgenic Mouse Production. In Lipoproteins and Cardiovascular Disease (L. A. Freeman, ed.) pp. 217‐232. Humana Press, Totowa, NJ Available at: http://link.springer.com/10.1007/978‐1‐60327‐369‐5_10.
  Quadros, R. M., Harms, D. W., Ohtsuka, M., and Gurumurthy, C. B. 2015. Insertion of sequences at the original provirus integration site of mouse ROSA26 locus using the CRISPR/Cas9 system. FEBS Open Bio 5:191‐197. doi: 10.1016/j.fob.2015.03.003.
  Sato, M., Akasaka, E., Saitoh, I., Ohtsuka, M., and Watanabe, S. 2012. In vivo gene transfer in mouse preimplantation embryos after intraoviductal injection of plasmid DNA and subsequent in vivo electroporation. Syst. Biol. Reprod. Med. 58:278‐287. doi: 10.3109/19396368.2012.688088.
  Skarnes, W. C. 2015. Is mouse embryonic stem cell technology obsolete? Genome Biol. 16. Available at: http://genomebiology.com/2015/16/1/109. doi: 10.1186/s13059-015-0673-6.
  Takahashi, G., Gurumurthy, C. B., Wada, K., Miura, H., Sato, M., and Ohtsuka, M. 2015. GONAD: Genome‐editing via Oviductal Nucleic Acids Delivery system: A novel microinjection independent genome engineering method in mice. Sci. Rep. 5:11406. doi: 10.1038/srep11406.
  Williams, E., Auerbach, W., DeChiara, T. M., and Gertsenstein, M. 2011. Combining ES Cells with Embryos. In Advanced Protocols for Animal Transgenesis (S. Pease and T. L. Saunders, eds.) pp. 377‐430. Springer Berlin Heidelberg, Berlin, Heidelberg available at: http://link.springer.com/10.1007/978‐3‐642‐20792‐1_17
Key References
  Harms et al., 2014. See above.
  This article describes the various steps involved in designing and producing genetically engineered mouse models using the CRISPR/Cas9 system. Many of the steps described in Harms et al are necessary to prepare reagents needed for the GONAD method.
  Takahashi et al., 2015. See above.
  This article is the first report of GONAD method description. It describes the scientific rationale for developing the method and also it includes several sets of data related to the first GONAD experiments.
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