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

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

Author information

1
Program in Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada.
2
Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.

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.

PMID:
29889212
DOI:
10.1038/nbt.4166
[Indexed for MEDLINE]

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