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

Bin Gu, Eszter Posfai, Janet Rossant

Research output: Contribution to journalArticlepeer-review

197 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)632-637
Number of pages6
JournalNature biotechnology
Issue number7
StatePublished - Aug 1 2018
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Applied Microbiology and Biotechnology
  • Bioengineering
  • Molecular Medicine
  • Biotechnology
  • Biomedical Engineering


Dive into the research topics of 'Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos'. Together they form a unique fingerprint.

Cite this