Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Creation of CRISPR-based germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD


Methods to create genetically engineered mice involve three major steps: harvesting embryos from one set of females, microinjection of reagents into embryos ex vivo and their surgical transfer to another set of females. Although tedious, these methods have been used for more than three decades to create mouse models. We recently developed a method named GONAD (genome editing via oviductal nucleic acids delivery), which bypasses these steps. GONAD involves injection of CRISPR components (Cas9 mRNA and guide RNA (gRNA)) into the oviducts of pregnant females 1.5 d post conception, followed by in vivo electroporation to deliver the components into the zygotes in situ. Using GONAD, we demonstrated that target genes can be disrupted and analyzed at different stages of mouse embryonic development. Subsequently, we developed improved GONAD (i-GONAD) by delivering CRISPR ribonucleoproteins (RNPs; Cas9 protein or Cpf1 protein and gRNA) into day-0.7 pregnant mice, which made it suitable for routine generation of knockout and large-deletion mouse models. i-GONAD can also generate knock-in models containing up to 1-kb inserts when single-stranded DNA (ssDNA) repair templates are supplied. i-GONAD offers other advantages: it does not require vasectomized males and pseudo-pregnant females, the females used for i-GONAD are not sacrificed and can be used for other experiments, it can be easily adopted in laboratories lacking sophisticated microinjection equipment, and can be implemented by researchers skilled in small-animal surgery but lacking embryo-handling skills. Here, we provide a step-by-step protocol for establishing the i-GONAD method. The protocol takes 6 weeks to generate the founder mice.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Overview of the i-GONAD workflow.
Fig. 2: Equipment and instruments required for i-GONAD procedure.
Fig. 3: Examples of 1% (wt/vol) agarose gels showing electrophoresis.
Fig. 4: Diagrams showing the surgical steps to expose ovary/oviduct/uterus (Steps 10–16).
Fig. 5: Diagrams showing loading of genome-editing reagents into an injection needle (Steps 17 and 18).
Fig. 6: Diagrams showing intraoviductal injection (Step 20).
Fig. 7: Diagrams showing in vivo electroporation (Steps 22–26).
Fig. 8: Diagrams showing steps after in vivo electroporation (Steps 27–30).

Similar content being viewed by others

Data availability

All the data generated and analyzed in this study are included in the tables, figures and supplementary material.


  1. Gordon, J. & Ruddle, F. Integration and stable germ line transmission of genes injected into mouse pronuclei. Science 214, 1244–1246 (1981).

    Article  CAS  Google Scholar 

  2. Evans, M. J. & Kaufman, M. H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981).

    Article  CAS  Google Scholar 

  3. Smithies, O., Gregg, R. G., Boggs, S. S., Koralewski, M. A. & Kucherlapati, R. S. Insertion of DNA sequences into the human chromosomal β-globin locus by homologous recombination. Nature 317, 230–234 (1985).

    Article  CAS  Google Scholar 

  4. Thomas, K. High frequency targeting of genes to specific sites in the mammalian genome. Cell 44, 419–428 (1986).

    Article  CAS  Google Scholar 

  5. Doyle, A., McGarry, M. P., Lee, N. A. & Lee, J. J. The construction of transgenic and gene knockout/knockin mouse models of human disease. Transgenic Res. 21, 327–349 (2012).

    Article  CAS  Google Scholar 

  6. Skarnes, W. C. et al. A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474, 337–342 (2011).

    Article  CAS  Google Scholar 

  7. Geurts, A. M. et al. Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325, 433 (2009).

    Article  CAS  Google Scholar 

  8. Bogdanove, A. J. & Voytas, D. F. TAL effectors: customizable proteins for DNA targeting. Science 333, 1843–1846 (2011).

    Article  CAS  Google Scholar 

  9. Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

    Article  CAS  Google Scholar 

  10. Gurumurthy, C. B. et al. GONAD: a novel CRISPR/Cas9 genome editing method that does not require ex vivo handling of embryos. Curr. Protoc. Hum. Genet. 88, 15.8 (2016).

  11. Harms, D. W. et al. Mouse genome editing using the CRISPR/Cas system. Curr. Protoc. Hum. Genet. 83, 15.7 (2014).

    Google Scholar 

  12. Teixeira, M. et al. Electroporation of mice zygotes with dual guide RNA/Cas9 complexes for simple and efficient cloning-free genome editing. Sci. Rep. 8, 474 (2018).

    Article  Google Scholar 

  13. Tröder, S. E. et al. An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes. PLoS ONE 13, e0196891 (2018).

    Article  Google Scholar 

  14. Chen, S., Lee, B., Lee, A. Y. F., Modzelewski, A. J. & He, L. Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes. J. Biol. Chem. 291, 14457–14467 (2016).

    Article  CAS  Google Scholar 

  15. Qin, W. et al. Efficient CRISPR/cas9-mediated genome editing in mice by zygote electroporation of nuclease. Genetics 200, 423–430 (2015).

    Article  CAS  Google Scholar 

  16. Hashimoto, M., Yamashita, Y. & Takemoto, T. Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse. Dev. Biol. 418, 1–9 (2016).

    Article  CAS  Google Scholar 

  17. Takahashi, G. et al. GONAD: Genome-editing via Oviductal Nucleic Acids Delivery system: a novel microinjection independent genome engineering method in mice. Sci. Rep. 5, 11406 (2015).

    Article  Google Scholar 

  18. Ohtsuka, M. et al. i-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases. Genome Biol. 19, 25 (2018).

    Article  Google Scholar 

  19. Ohtsuka, M. et al. Pronuclear injection-based mouse targeted transgenesis for reproducible and highly efficient transgene expression. Nucleic Acids Res. 38, e198 (2010).

    Article  Google Scholar 

  20. Behringer, R., Gertsenstein, M., Nagy, K. V. & Nagy, A. Manipulating the Mouse Embryo: A Laboratory Manual, 4th edn. (Cold Spring Harbor Laboratory Press, 2014).

  21. Quadros, R. M. et al. Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Genome Biol. 18, 92 (2017).

    Article  Google Scholar 

  22. Miura, H., Gurumurthy, C. B., Sato, T., Sato, M. & Ohtsuka, M. CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA. Sci. Rep. 5, 12799 (2015).

    Article  CAS  Google Scholar 

  23. Miura, H., Quadros, R. M., Gurumurthy, C. B. & Ohtsuka, M. Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors. Nat. Protoc. 13, 195–215 (2018).

    Article  CAS  Google Scholar 

  24. Kobayashi, T. et al. Successful production of genome-edited rats by the rGONAD method. BMC Biotechnol. 18, 19 (2018).

    Article  Google Scholar 

  25. Takabayashi, S. et al. i-GONAD (improved genome-editing via oviductal nucleic acids delivery), a convenient in vivo tool to produce genome-edited rats. Sci. Rep. 8, 12059 (2018).

    Article  Google Scholar 

  26. Miano, J. M., Zhu, Q. M. & Lowenstein, C. J. A CRISPR path to engineering new genetic mouse models for cardiovascular research. Arterioscler. Thromb. Vasc. Biol. 36, 1058–1075 (2016).

    Article  CAS  Google Scholar 

  27. Haeussler, M. et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol. 17, 148 (2016).

    Article  Google Scholar 

  28. Labun, K., Montague, T. G., Gagnon, J. A., Thyme, S. B. & Valen, E. CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Res. 44, W272–W276 (2016).

    Article  CAS  Google Scholar 

  29. Oliveros, J. C. et al. Breaking-Cas—interactive design of guide RNAs for CRISPR-Cas experiments for ENSEMBL genomes. Nucleic Acids Res. 44, W267–W271 (2016).

    Article  CAS  Google Scholar 

  30. Aida, T. et al. Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice. Genome Biol. 16, 87 (2015).

    Article  Google Scholar 

  31. Morrow, D. A. Prognostic value of serial B-type natriuretic peptide testing during follow-up of patients with unstable coronary artery disease. JAMA 294, 2866–2871 (2005).

    Article  CAS  Google Scholar 

  32. Ma, H. & Difazio, S. An efficient method for purification of PCR products for sequencing. Biotechniques 44, 921–923 (2008).

    Article  CAS  Google Scholar 

  33. Dinkel, A. et al. Efficient generation of transgenic BALB/c mice using BALB/c embryonic stem cells. J. Immunol. Methods 223, 255–260 (1999).

    Article  CAS  Google Scholar 

  34. Dong, L., Lv, L. B. & Lai, R. [Molecular cloning of Tupaia belangeri chinensis neuropeptide Y and homology comparison with other analogues from primates]. Zool. Res. 33, 75–78 (2012).

    Article  CAS  Google Scholar 

Download references


We thank Y. Ishikawa (Tokai University) for preparing pregnant MCH(ICR) mice and BEX Co. Ltd for advising us about electroporation optimization. This work was supported by the 2014 Tokai University School of Medicine Research Aid, the Research and Study Project of Tokai University General Research Organization, the 2016–2017 Tokai University School of Medicine Project Research, the Research Aid from the Institute of Medical Sciences inTokai University, the MEXT‐Supported Program for the Strategic Research Foundation at Private Universities 2015–2019, and a Grant-in-Aid for Challenging Exploratory Research (15K14371) from JSPS to M.O. AsCpf1 protein was a gift fromIDT. We thank J. M. Miano (University of Rochester) and G. Burgio (Australian National University) for their helpful comments on the manuscript.

Author information

Authors and Affiliations



M.O., C.B.G. and M.S. conceived the idea of i-GONAD. M.O., M.S. and M.A.I. were involved in testing the 2-d protocol. H.M. established the long ssDNA preparation system. M.I. and N.K. tested the hand-made capillary-based i-GONAD method. M.O., A.N. and S.O. optimized i-GONAD in the C57BL/6 strain. S.N. tested the squeezing steps in the i-GONAD procedure. M.O., S.T. and M.M. performed and optimized i-GONAD in several mouse strains and electroporators. C.B.G., M.S., M.I., M.A.I., S.N., H.M. and M.O. contributed to the writing of the manuscript.

Corresponding authors

Correspondence to Channabasavaiah B. Gurumurthy or Masato Ohtsuka.

Ethics declarations

Competing interests

C.B.G., M.S. and M.O. have filed a patent application relating to the work described in this paper with International Patent Application no. PCT/US2018/047748, filed August 23, 2017 (Methods and compositions for in situ germline genome engineering). Tokai University and BEX Co. Ltd. applied for a patent describing the electroporation condition using CUY21Edit II on application number 2017–233100 (filed December 5, 2017). M.O. is an inventor of the patent.

Additional information

Peer review information: Nature Protocols thanks Izuho Hatada, Michael Wiles and other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Ohtsuka, M. et al. Genome Biol. 19, 25 (2018):

Takahashi, G. et al. Sci. Rep. 5, 11406 (2015):

Miura, H., Quadros, R. M., Gurumurthy, C. B. & Ohtsuka, M. Nat. Protoc. 13, 195–215 (2018):

Integrated supplementary information

Supplementary Fig. 1 Preparation of devises used for intraoviductal injection.

(a) Cartoon showing various parts of a mouth pipetting devise. (b) Cartoon showing glass capillaries needed for intraoviductal injection. Drawings of a capillary tip before and after it was cut with the microscissors.

Supplementary Fig. 2 Dissecting microscopic view of oviduct after injection of solution containing Fast Green FCF.

Images of the oviduct photographed immediately after injection (a), and after covering it with a wet Kimwipe towel (b).

Supplementary Fig. 3 Genome-editing efficiency and number of fetuses retrieved after performing i-GONAD with different electroporation currents.

Tyr rescue experiment is shown as an example. Two MCH(ICR) females were used for each current value. Gray bar indicates a total number of mid-gestation fetuses recovered from two females. Yellow bar indicates a total number of genome modified fetuses (includes both knock-in and indel). Blue bar indicates a total number of fetuses with pigmentation (successful knock-in). Green line indicates the % of genome edited fetuses. The data of this experiment are available from the corresponding author upon request.

Supplementary Fig. 4 Injection of solution using hand-made glass capillary.

(a) Dissecting microscopic view of injection (see also Supplementary Video 3). (b) Tip of hand-made glass capillary.

Supplementary Fig. 5 Cartoons showing collection of 2-cell-stage embryos from the i-GONAD-treated mouse (procedure 1 of 2).

(a) Making a midline incision at the ventral surface of the euthanized mouse. (b) Opening the ventral skin. (c) Opening the ventral muscle. (d) Pulling out of both ovaries/oviducts/uteri and trimming off the portion of the uterus. (e) Placing of the dissected ovaries/oviducts/uteri and adding a small amount of 1x PBS onto the tissues. (f) Removing of the associated blood on ovary/oviduct/uterus by placing them onto a paper towel. (g) Removing the mesenterium (a membranous fold) associated with the uterus and oviduct. (h) Removing the adipose tissue associated with an ovary. (i) Removing the ovary using microscissors under a dissecting microscope. R, right; L, left.

Supplementary Fig. 6 Cartoons showing collection of 2-cell-stage embryos from the i-GONAD-treated mouse (procedure 2 of 2).

(a) and (b) Cutting of the junctional portion between oviduct and uterus using microscissors. (c) Inserting the 30-G needle attached with a 1-ml disposable plastic syringe into the oviductal lumen. (d) Flushing the contents while watching the flowing out of embryos. (e) Removing the oviduct and collecting the released embryos using an egg handling pipette. The picture above the drops (top right) is the side view of the embryo handling procedure in the drop. Washing of the collected embryos by passing them through small drops, and (f) transferring them into another fresh drop ready for observing under an inverted fluorescence microscope. Embryos obtained from four females (#1 to #4) are shown as an example. R, embryos from right oviduct; L, embryos from left oviduct. Right most image shows a side view of microscopic observation.

Supplementary Fig. 7 Fluorescence in the preimplantation mouse embryos after i-GONAD.

(a) - (c) Two-cell embryos collected from the MCH(ICR) females that were subjected to i-GONAD procedure following injection of a solution containing tetramethylrhodamine-labeled dextran 3kDa (red), EGFP mRNA (green). Note that the embryos are observed for both red and green fluorescence. (d) to (f) Two-cell embryos collected from the MCH(ICR) females similarly treated except the electroporation step showing no fluorescence in any of the embryos. Scale bar = 100 µM.

Supplementary information

Supplementary Information

Supplementary Figs. 1–7

Reporting Summary

Supplementary Video 1

Surgical exposure of an oviduct.

Supplementary Video 2

Loading solution into a capillary needle.

Supplementary Video 3

Injecting solution into an oviduct by using a hand-made capillary with injection choice 1 (Fig. 6).

Supplementary Video 4

Injecting solution into an oviduct and then performing electroporation on the oviduct with an NEPA21 electroporator and CUY652-3 electrodes (2-d protocol) using injection choice 1 (Fig. 6).

Supplementary Video 5

Squeezing of the ampulla to disperse the injected solution uniformly within the lumen.

Supplementary Video 6

Injecting solution into an oviduct and then performing electroporation on the oviduct with a CUY21EditII electroporator and LF650P3 electrodes using injection choice 1 (Fig. 6).

Supplementary Video 7

Preparing a glass micropipette with a flame.

Supplementary Video 8

Cutting the tip of a glass capillary.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gurumurthy, C.B., Sato, M., Nakamura, A. et al. Creation of CRISPR-based germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD. Nat Protoc 14, 2452–2482 (2019).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing