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

Nat Biotechnol. 2018 Aug;36(7):632-637. doi: 10.1038/nbt.4166. Epub 2018 Jun 11.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacterial Proteins / genetics
  • Biotin / analogs & derivatives
  • Biotin / genetics
  • Blastocyst
  • CRISPR-Cas Systems / genetics*
  • DNA Repair / genetics
  • Gene Editing / methods*
  • Gene Expression Regulation, Developmental
  • Gene Knock-In Techniques
  • Gene Transfer Techniques*
  • Genome / genetics*
  • Homologous Recombination / genetics
  • Mice


  • Bacterial Proteins
  • biotin-streptavidin complex
  • Biotin