Optimization of the one-step CRISPR/Cas9 gene knockout employing reporter transgenic zygotes

BEVACQUA RJ; RONJA APFELBAUM; GARRELS W; TALLURI TR; MUKHERJEE A; ZIEGLER M; BURCHARDT B; SALAMONE D; NIEMANN H; GRUESO E; IVICS Z; KUES W

Congreso; 8th International Meeting of Stem Cell Network North Rhine Westphalia; 2015

The injection of CRISPR/Cas9 components directly into one-cell embryos allows genome editing in animals in a one-step manner. Here, we present a fluorophore mouse model for establishment and optimization of this one-step genome-editing tool. The model is based on a transgenic mouse line, carrying a monomeric CAGGS-Venus Sleeping Beauty transposon. The CAGGS promoter driven Venus fluorophore is systemically expressed, and Venus expression is not affected by epigenetic or environmental factors. Here, the cytoplasmic injection of Cas9 and sgRNA plasmids into Venus zygotes was assessed (recovered zygotes: 43; injected zygotes: 38; transferred zygotes: 34) and knock-out of the Venus reporter was achieved in 28% of the born pups. Importantly, the majority of knock-out pups showed a complete knock-out in all organs. The model allows the direct comparison of different compositions of the injections solution (plasmids, RNAs, ribonucleotide/Cas9 complexes), different injections sites (pronuclear, cytoplasmic), as well as the optimization of other parameters (time point of injection, injection volume, concentration). Depending on the breeding schedule, the efficacy of mono- and bi-allelic knock-outs can be assessed.