Single-Step Generation of Conditional Knockout Mouse Embryonic Stem Cells

Cell Rep. 2015 Jul 28;12(4):709-16. doi: 10.1016/j.celrep.2015.06.051. Epub 2015 Jul 16.


Induction of double-strand DNA breaks (DSBs) by engineered nucleases, such as CRISPR/Cas9 or transcription activator-like effector nucleases (TALENs), stimulates knockin of exogenous DNA fragments via homologous recombination (HR). However, the knockin efficiencies reported so far have not allowed more complex in vitro genome modifications such as, for instance, simultaneous integration of a DNA fragment at two distinct genomic sites. We developed a reporter system to enrich for cells with engineered nuclease-assisted HR events. Using this system in mouse embryonic stem cells (mESCs), we achieve single-step biallelic and seamless integration of two loxP sites for Cre recombinase-mediated inducible gene knockout, as well as biallelic endogenous gene tagging with high efficiency. Our approach reduces the time and resources required for conditional knockout mESC generation dramatically.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cells, Cultured
  • Embryonic Stem Cells / cytology*
  • Embryonic Stem Cells / metabolism
  • Gene Knockout Techniques / methods*
  • Homologous Recombination
  • Integrases / genetics
  • Integrases / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Tissue Culture Techniques / methods*


  • Cre recombinase
  • Integrases