Engineering of the mouse germline to create targeted mutants is a key technology for biomedical research. We use an expedite approach for the generation of mouse mutants by microinjection of engineered, sequence-specific nucleases into one-cell embryos. Such nucleases create targeted double-strand breaks (DSBs) and stimulate DNA repair by non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ religates the open ends, frequently leading to frameshift (knockout) mutations by the loss of nucleotides, whereas HDR enables the insertion of targeted (knockin) mutations from gene targeting vectors or oligonucleotides as repair templates. By this means mutant knockout and knockin founders are identified 7 weeks after embryo injections, enabling the fast establishment of mutant lines. Three nuclease generations, ZFNs, TALENs and the CRISPR/Cas9 system were validated in recent years for direct mutagenesis in embryos. In particular, CRISPR/Cas9 enables the generation of knockout and knockin alleles at frequencies of up to 40% and 10%, respectively, among pups derived from embryo injections. Nevertheless, the dominance of NHEJ versus HDR requires further improvement. To tackle this problem we established `traffic light´ reporter lines indicating DSB repair by NHEJ or HDR through the expression of red or green fluorescent proteins. To enhance HDR, we suppressed NHEJ key molecules by gene silencing, by the inhibitor SCR7 or by the adenoviral proteins E1B55K and E4orf6. In cell lines, SCR7 or the knockdown of KU70 and DNA Ligase IV promotes the efficiency of HDR up to 5-fold. Coexpression of the DNA Ligase IV degrading E1B55K and E4orf6 proteins improves the efficiency of HDR up to 8-fold and essentially abolishes NHEJ repair. We are presently using TLR transgenic mice to enhance HDR repair of CRISPR/Cas-induced DSBs by NHEJ suppression in early embryos and somatic cells to optimize the generation of precisely targeted alleles in vivo.
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