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

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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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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.


  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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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.

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    Supplementary Text and Figures

    Supplementary Figures 1–10 and Supplementary Table 1

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    Life Sciences Reporting Summary

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    Supplementary Table 2

    Oligo sequences


  1. 1.

    Video of microinjecting mouse 2-cell embryos

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    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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