Letter | Published:

Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos

Nature Biotechnology volume 36, pages 632637 (2018) | Download Citation

Abstract

Rapid, efficient generation of knock-in mice with targeted large insertions remains a major hurdle in mouse genetics. Here, we describe two-cell homologous recombination (2C-HR)-CRISPR, a highly efficient gene-editing method based on introducing CRISPR reagents into embryos at the two-cell stage, which takes advantage of the open chromatin structure and the likely increase in homologous-recombination efficiency during the long G2 phase. Combining 2C-HR-CRISPR with a modified biotin–streptavidin approach to localize repair templates to target sites, we achieved a more-than-tenfold increase (up to 95%) in knock-in efficiency over standard methods. We targeted 20 endogenous genes expressed in blastocysts with fluorescent reporters and generated reporter mouse lines. We also generated triple-color blastocysts with all three lineages differentially labeled, as well as embryos carrying the two-component auxin-inducible degradation system for probing protein function. We suggest that 2C-HR-CRISPR is superior to random transgenesis or standard genome-editing protocols, because it ensures highly efficient insertions at endogenous loci and defined 'safe harbor' sites.

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References

  1. 1.

    Genome engineering with targetable nucleases. Annu. Rev. Biochem. 83, 409–439 (2014).

  2. 2.

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

  3. 3.

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

  4. 4.

    et al. Homology-mediated end joining-based targeted integration using CRISPR/Cas9. Cell Res. 27, 801–814 (2017).

  5. 5.

    'Any idiot can do it': genome editor CRISPR could put mutant mice in everyone's reach. Science (2016).

  6. 6.

    et al. Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes. BMC Biotechnol. 16, 4 (2016).

  7. 7.

    et al. Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9. Nat. Commun. 5, 5560 (2014).

  8. 8.

    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).

  9. 9.

    et al. RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency. Nat. Commun. 7, 10548 (2016).

  10. 10.

    et al. Efficient generation of mice carrying homozygous double-floxp alleles using the Cas9-Avidin/Biotin-donor DNA system. Cell Res. 27, 578–581 (2017).

  11. 11.

    et al. Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering. eLife 6, e25312 (2017).

  12. 12.

    & Making the blastocyst: lessons from the mouse. J. Clin. Invest. 120, 995–1003 (2010).

  13. 13.

    et al. Single-molecule dynamics of enhanceosome assembly in embryonic stem cells. Cell 156, 1274–1285 (2014).

  14. 14.

    et al. Spatio-temporally precise activation of engineered receptor tyrosine kinases by light. EMBO J. 33, 1713–1726 (2014).

  15. 15.

    , , , & An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat. Methods 6, 917–922 (2009).

  16. 16.

    & The control of DNA repair by the cell cycle. Nat. Cell Biol. 19, 1–9 (2016).

  17. 17.

    et al. Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases. Sci. Rep. 6, 21264 (2016).

  18. 18.

    , , & Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife 3, e04766 (2014).

  19. 19.

    , , , & Post-translational regulation of Cas9 during G1 enhances homology-directed repair. Cell Reports 14, 1555–1566 (2016).

  20. 20.

    , , & Rapid protein depletion in human cells by auxin-inducible degron tagging with short homology donors. Cell Reports 15, 210–218 (2016).

  21. 21.

    & The HaloTag: a novel technology for cell imaging and protein analysis. Methods Mol. Biol. 356, 195–208 (2007).

  22. 22.

    & Development of 2A peptide-based strategies in the design of multicistronic vectors. Expert Opin. Biol. Ther. 5, 627–638 (2005).

  23. 23.

    et al. Bright monomeric near-infrared fluorescent proteins as tags and biosensors for multiscale imaging. Nat. Commun. 7, 12405 (2016).

  24. 24.

    et al. Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst. Dev. Cell 18, 675–685 (2010).

  25. 25.

    et al. Position- and Hippo signaling-dependent plasticity during lineage segregation in the early mouse embryo. eLife 6, e22906 (2017).

  26. 26.

    et al. Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells. Proc. Natl. Acad. Sci. USA 94, 3789–3794 (1997).

  27. 27.

    , , & Stable, high-affinity streptavidin monomer for protein labeling and monovalent biotin detection. Biotechnol. Bioeng. 110, 57–67 (2013).

  28. 28.

    et al. Genome editing reveals a role for OCT4 in human embryogenesis. Nature 550, 67–73 (2017).

  29. 29.

    & Generation and live imaging of an endogenous Cdx2 reporter mouse line. Genesis 50, 775–782 (2012).

  30. 30.

    et al. Inverted light-sheet microscope for imaging mouse pre-implantation development. Nat. Methods 13, 139–142 (2016).

  31. 31.

    et al. Adaptive light-sheet microscopy for long-term, high-resolution imaging in living organisms. Nat. Biotechnol. 34, 1267–1278 (2016).

  32. 32.

    , , & The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans. Development 142, 4374–4384 (2015).

  33. 33.

    , , , & AIRE is a critical spindle-associated protein in embryonic stem cells. eLife 6, e28131 (2017).

  34. 34.

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

  35. 35.

    et al. Following cell fate in the living mouse embryo. Development 124, 1133–1137 (1997).

  36. 36.

    et al. A general method to improve fluorophores for live-cell and single-molecule microscopy. Nat. Methods 12, 244–250 (2015).

  37. 37.

    , , & The Hippo pathway member Nf2 is required for inner cell mass specification. Curr. Biol. 23, 1195–1201 (2013).

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Acknowledgements

The authors acknowledge technical support from the Model Production Core staff led by M. Gertsenstein at the Centre for Phenogenomics; L. Lavis (HHMI Janelia Research Campus) for synthetic dyes against the Halo-tag; D. Durocher (Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital) for discussion of the parameters affecting HR; Y. Yamanaka (McGill Cancer Research Center) for previously setting up the two-cell microinjection system in the Rossant laboratory; and S. Park (University at Buffalo), R. Kuehn (Berlin Institute of Health) and F. Zhang (Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard) for providing materials. This work was funded by CIHR (FDN-143334) to J.R. and Genome Canada and Ontario Genomics (OGI-099).

Author information

Author notes

    • Bin Gu
    •  & Eszter Posfai

    These authors contributed equally to this work.

Affiliations

  1. Program in Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada.

    • Bin Gu
    • , Eszter Posfai
    •  & Janet Rossant
  2. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.

    • Janet Rossant

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Contributions

B.G., E.P. and J.R. conceived the study. B.G. conceived increasing HR by microinjecting two-cell embryos. B.G. and E.P. designed, carried out and analyzed all experiments equally. J.R. provided supervision and funding for the study. All authors contributed to writing the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Janet Rossant.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–10 and Supplementary Table 1

  2. 2.

    Life Sciences Reporting Summary

Excel files

  1. 1.

    Supplementary Table 2

    Oligo sequences

Videos

  1. 1.

    Video of microinjecting mouse 2-cell embryos

  2. 2.

    Live imaging of Gata6-Halo/Cdx2-eGFP mouse embryos from 16-cell stage to blastocyst

    Live imaging of mouse embryos carrying primitive endoderm reporter Gata6-Halo and Trophectoderm reporter Cdx2-eGFP for 30 hours 16-cell stage to blastocyst

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DOI

https://doi.org/10.1038/nbt.4166