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.

Generating genetically modified mice using CRISPR/Cas-mediated genome engineering


Mice with specific gene modifications are valuable tools for studying development and disease. Traditional gene targeting in mice using embryonic stem (ES) cells, although suitable for generating sophisticated genetic modifications in endogenous genes, is complex and time-consuming. We have recently described CRISPR/Cas-mediated genome engineering for the generation of mice carrying mutations in multiple genes, endogenous reporters, conditional alleles or defined deletions. Here we provide a detailed protocol for embryo manipulation by piezo-driven injection of nucleic acids into the cytoplasm to create gene-modified mice. Beginning with target design, the generation of gene-modified mice can be achieved in as little as 4 weeks. We also describe the application of the CRISPR/Cas technology for the simultaneous editing of multiple genes (five genes or more) after a single transfection of ES cells. The principles described in this protocol have already been applied in rats and primates, and they are applicable to sophisticated genome engineering in species in which ES cells are not available.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: sgRNA and donor DNA construction.
Figure 2: Procedure for the generation of gene-modified mice by CRISPR/Cas.
Figure 3: Schematic illustrating HDR-mediated gene editing by an ssDNA template at a DSB created by Cas9.
Figure 4: Suggested layout of droplets in the manipulation chamber.
Figure 5: Injection of zygotes.


  1. Capecchi, M.R. Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat. Rev. Genet. 6, 507–512 (2005).

    CAS  Article  Google Scholar 

  2. Carbery, I.D. et al. Targeted genome modification in mice using zinc-finger nucleases. Genetics 186, 451–459 (2010).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  4. Brown, A.J. et al. Whole-rat conditional gene knockout via genome editing. Nat. Methods 10, 638–640 (2013).

    CAS  Article  Google Scholar 

  5. Cui, X. et al. Targeted integration in rat and mouse embryos with zinc-finger nucleases. Nat. Biotechnol. 29, 64–67 (2011).

    CAS  Article  Google Scholar 

  6. Sung, Y.H. et al. Knockout mice created by TALEN-mediated gene targeting. Nat. Biotechnol. 31, 23–24 (2013).

    CAS  Article  Google Scholar 

  7. Tesson, L. et al. Knockout rats generated by embryo microinjection of TALENs. Nat. Biotechnol. 29, 695–696 (2011).

    CAS  Article  Google Scholar 

  8. Carlson, D.F. et al. Efficient TALEN-mediated gene knockout in livestock. Proc. Natl. Acad. Sci. USA 109, 17382–17387 (2012).

    CAS  Article  Google Scholar 

  9. Shen, B. et al. Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res. 23, 720–723 (2013).

    CAS  Article  Google Scholar 

  10. Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).

    CAS  Article  Google Scholar 

  11. Yang, H. et al. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154, 1370–1379 (2013).

    CAS  Article  Google Scholar 

  12. Li, W., Teng, F., Li, T. & Zhou, Q. Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems. Nat. Biotechnol. 31, 684–686 (2013).

    CAS  Article  Google Scholar 

  13. Li, D. et al. Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat. Biotechnol. 31, 681–683 (2013).

    CAS  Article  Google Scholar 

  14. Hwang, W.Y. et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat. Biotechnol. 31, 227–229 (2013).

    CAS  Article  Google Scholar 

  15. Niu, Y. et al. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 156, 836–843 (2014).

    CAS  Article  Google Scholar 

  16. Meyer, M., de Angelis, M.H., Wurst, W. & Kuhn, R. Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases. Proc. Natl. Acad. Sci. USA 107, 15022–15026 (2010).

    CAS  Article  Google Scholar 

  17. Horvath, P. & Barrangou, R. CRISPR/Cas, the immune system of bacteria and archaea. Science 327, 167–170 (2010).

    CAS  Article  Google Scholar 

  18. Wiedenheft, B., Sternberg, S.H. & Doudna, J.A. RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331–338 (2012).

    CAS  Article  Google Scholar 

  19. Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl. Acad. Sci. USA 109, E2579–E2586 (2012).

    CAS  Article  Google Scholar 

  20. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    CAS  Article  Google Scholar 

  21. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    CAS  Article  Google Scholar 

  22. Esvelt, K.M. et al. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat. Methods 10, 1116–1121 (2013).

    CAS  Article  Google Scholar 

  23. Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826 (2013).

    CAS  Article  Google Scholar 

  24. Hsu, P.D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

    CAS  Article  Google Scholar 

  25. Ran, F.A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    CAS  Article  Google Scholar 

  26. Ittner, L.M. & Gotz, J. Pronuclear injection for the production of transgenic mice. Nat. Protoc. 2, 1206–1215 (2007).

    CAS  Article  Google Scholar 

  27. Eggan, K. et al. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc. Natl. Acad. Sci. USA 98, 6209–6214 (2001).

    CAS  Article  Google Scholar 

  28. Tucker, K.L., Wang, Y., Dausman, J. & Jaenisch, R. A transgenic mouse strain expressing four drug-selectable marker genes. Nucleic Acids Res. 25, 3745–3746 (1997).

    CAS  Article  Google Scholar 

  29. Markoulaki, S., Meissner, A. & Jaenisch, R. Somatic cell nuclear transfer and derivation of embryonic stem cells in the mouse. Methods 45, 101–114 (2008).

    CAS  Article  Google Scholar 

  30. Guschin, D.Y. et al. A rapid and general assay for monitoring endogenous gene modification. Methods Mol. Biol. 649, 247–256 (2010).

    CAS  Article  Google Scholar 

Download references


This work was supported by US National Institutes of Health (NIH) grants R37-HD045022 and R01-CA084198. This work was also supported by a grant from the Simons Foundation to the Simons Center for the Social Brain at the Massachusetts Institute of Technology.

Author information

Authors and Affiliations



H.Y., H.W. and R.J. wrote the manuscript.

Corresponding author

Correspondence to Rudolf Jaenisch.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Injection of CRISPR/Cas mix into zygotes by piezo-based cytoplasmic injection.

The zygote is held by holding pipette on the left side and then injected with CRISPR/Cas mix by injection pipette on the right side. All the injection steps are carried out in CB medium (also described in Fig. 4). (MP4 14163 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Yang, H., Wang, H. & Jaenisch, R. Generating genetically modified mice using CRISPR/Cas-mediated genome engineering. Nat Protoc 9, 1956–1968 (2014).

Download citation

  • Published:

  • Issue Date:

  • DOI:

Further reading


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