Cas9-assisted mouse embryonic stem cell targeting

In a partnership with the Transgenic Core at the Wellcome Trust Centre for Human Genetics we combine our expertise in generating donor constructs for Cas9-assisted homologous recombination with their mouse ESC targeting pipeline for the generation of genetically modified mouse models. We refer to this service as development of a “full mouse package”.

Gene targeting in ES cells mediated by homologous recombination was awarded the Nobel Prize in 2007. Any targeted DNA sequence can be deleted constitutively or conditionally (KO and cKO), and specific mutations or expression cassettes can be inserted precisely within a region of interest (KI and cKI approaches). Until the advent of the CRISPR/Cas9 technology, targeting had to rely on linearized DNA donors bearing negative and positive selection markers. Screening of correctly targeted clones had to discriminate random versus proper insertion by Southern hybridization, with success rates often lower than 1-5%. Using Cas9 to generate specific DSBs directly at the insertion site, we can use circular donors for the homologous recombination process, and thus greatly reduce random insertion events. In concert, the higher rate of proper HR allows to use shorter homology arms, making the first screening amenable for a PCR-based approach. Once confirmed, cells are expanded and injected into pre-implantation embryos. The resulting chimeras are used for establishing the genetically modified mouse line.

 

Services we offer (in collaboration with the WTCHG)

 

Services we offer (in collaboration with the WIMM TG Services)