Single-Step Generation of Conditional Knockout Mouse Embryonic Stem Cells

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. [Display omitted] •A recombination reporter efficiently enriches for TALEN-mediated HR events•ESC lines for generation of conditional knockouts and gene tagging are established•Marker-less conditional knockout ESCs are generated in a single transfection step Flemr and Bühler introduce a reporter system for enriching cells with engineered nuclease-mediated homologous recombination events. The reporter enables simultaneous biallelic integration of two single-stranded oligodeoxynucleotides carrying loxP sites in mouse embryonic stem cells to generate conditional knockouts in a single transfection step.