Genetically altered (GA) mouse models provide a powerful means of attributing function to DNA sequences. As investigations of how DNA variation and mutation contributes to disease advance, demand for GA mouse models increases. Recently, this demand has been aided by the new CRISPR/Cas9 technology, an RNA guided nuclease system, which permits the production of models at unprecedented efficiencies.
Already the biomedical community is applying this new technology widely for mouse model production. Meanwhile, the 3Rs impact of the technology remains unexplored. This proposal seeks to address aspects of the technology that, our experience shows, can lead to substantial increases in mouse usage and wasted production of undesirable genotypes.
Following CRISPR/Cas9 microinjection into mouse zygotes, the founders generated are invariably mosaic, due to the persistence of the nuclease after the first cleavage event. Founders must be bred extensively to ensure transmission of the desired allele, increasing mouse usage. The mosaicism also precludes the phenotyping of the founder generation, which, given the high efficiencies of CRISPR/Cas9 might be feasible for certain phenotypic screens.
We plan to explore whether the use of Cas9 fusions with destabilizing or cell-cycle regulatory domains can be used to confine nuclease activity to the 1-cell stage and thus eliminate mosaicism. Furthermore, the mode of Cas9 supply, protein or maternally contributed via transgenic Cas9 overexpression will be investigated for its impact on mosaicism. As a potential refinement, methods to address and limit deleterious indel mutagenesis during knock-in mice production, a class of model particularly sought after for interrogating disease-related mutation, will be investigated. Whether the mode of nuclease delivery, the use of cell cycle-regulated Cas9 and co-injection of wild-type repair templates can impact the rate of indel mutation at the non-targeted allele will be assessed.