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Genetically modified animals continue to provide important insights in biomedical sciences. Research has focused mostly on genetically modified mice so far, but other species like pigs resemble more closely the human physiology. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR/Cas9. Here, we report for the first time the generation of Cas9 transgenic chickens and pigs that allow in vivo genome editing in these two important agricultural species. We demonstrated that Cas9 is constitutively expressed in all organs of both species and that the animals are healthy and fertile. In addition, we confirmed the functionality of Cas9 for a number of different target genes and for a variety of cell types. Taken together, these transgenic animal species expressing Cas9 provide an unprecedented tool for agricultural and biomedical research, and will facilitate organ specific reverse genetics as well as cross-species comparisons.

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