Protocol | Published:

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

Nature Protocols volume 9, pages 19561968 (2014) | Download Citation

Abstract

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.

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References

  1. 1.

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

  2. 2.

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

  3. 3.

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

  4. 4.

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

  5. 5.

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

  6. 6.

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

  7. 7.

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

  8. 8.

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

  9. 9.

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

  10. 10.

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

  11. 11.

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

  12. 12.

    , , & Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems. Nat. Biotechnol. 31, 684–686 (2013).

  13. 13.

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

  14. 14.

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

  15. 15.

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

  16. 16.

    , , & Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases. Proc. Natl. Acad. Sci. USA 107, 15022–15026 (2010).

  17. 17.

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

  18. 18.

    , & RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331–338 (2012).

  19. 19.

    , , & Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl. Acad. Sci. USA 109, E2579–E2586 (2012).

  20. 20.

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

  21. 21.

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

  22. 22.

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

  23. 23.

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

  24. 24.

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

  25. 25.

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

  26. 26.

    & Pronuclear injection for the production of transgenic mice. Nat. Protoc. 2, 1206–1215 (2007).

  27. 27.

    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).

  28. 28.

    , , & A transgenic mouse strain expressing four drug-selectable marker genes. Nucleic Acids Res. 25, 3745–3746 (1997).

  29. 29.

    , & Somatic cell nuclear transfer and derivation of embryonic stem cells in the mouse. Methods 45, 101–114 (2008).

  30. 30.

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

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Acknowledgements

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

Affiliations

  1. Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.

    • Hui Yang
    • , Haoyi Wang
    •  & Rudolf Jaenisch
  2. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Rudolf Jaenisch

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Contributions

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

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Rudolf Jaenisch.

Supplementary information

Videos

  1. 1.

    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).

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DOI

https://doi.org/10.1038/nprot.2014.134

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