Genome editing technology provides new possibilities for animal breeding and aid in understanding host-pathogen interactions. In poultry, retroviruses display one of the most difficult pathogens to control by conventional strategies such as vaccinations. Avian leukosis virus subgroup J (ALV-J) is an oncogenic, immunosuppressive retrovirus that causes myeloid leukosis and other tumors in chickens. Severe economic losses caused by ALV-J remain an unsolved problem in many parts of the world due to inefficient eradication strategies and lack of effective vaccines. ALV-J attachment and entry are mediated through the specific receptor, chicken Na + /H + exchanger type 1 (chNHE1). The non-conserved amino acid tryptophan 38 (W38) in chNHE1 is crucial for virus entry, making it a favorable target for the introduction of disease resistance. In this study, we obtained ALV-J-resistance in a commercial chicken line by precise deletion of chNHE1 W38, utilizing the CRISPR/Cas9-system in combination with homology directed repair. The genetic modification completely protected cells from infection with a subgroup J retrovirus. W38 deletion did neither have a negative effect on the development nor on the general health condition of the gene edited chickens. Overall, the generation of ALV-J-resistant birds by precise gene editing demonstrates the immense potential of this approach as an alternative disease control strategy in poultry.

43Genetically modified animals continue to provide important insights in biomedical sciences. 44 Research has focused mostly on genetically modified mice so far, but other species like pigs 45 resemble more closely the human physiology. In addition, cross-species comparisons with 46 phylogenetically distant species such as chickens provide powerful insights into fundamental 47 biological and biomedical processes. One of the most versatile genetic methods applicable across 48 species is CRISPR/Cas9. Here, we report for the first time the generation of Cas9 transgenic 49 chickens and pigs that allow in vivo genome editing in these two important agricultural species. 50We demonstrated that Cas9 is constitutively expressed in all organs of both species and that the 51 animals are healthy and fertile. In addition, we confirmed the functionality of Cas9 for a number 52 of different target genes and for a variety of cell types. Taken together, these transgenic animal 53 species expressing Cas9 provide an unprecedented tool for agricultural and biomedical research, 54 and will facilitate organ specific reverse genetics as well as cross-species comparisons. 55Significance statement 56 Genome engineering of animals is crucial for translational medicine and the study of genetic traits. 57Here, we generated transgenic chickens and pigs that ubiquitously express the Cas9 endonuclease, 58 providing the basis for in vivo genome editing. We demonstrated the functionality of this system 59 3 by successful genome editing in chicken and porcine cells and tissues. These animals facilitate 60 organ specific in vivo genome editing in both species without laborious germ line modifications, 61 which will reduce the number of animals needed for genetic studies. They also provide a new tool 62 for functional genomics, developmental biology and numerous other applications in biomedical 63 and agricultural science.64 Introduction 65 Chickens and pigs are the most important livestock species worldwide. They are not only important 66 sources of food, but also valuable models for evolutionary biology and biomedical science. Pigs 67 share a high anatomical and physiological similarity with humans, and are an important species for 68 translational biomedical research e.g. in the areas of cancer, diabetes, neurodegenerative and 69 cardiovascular diseases (1-3). In contrast, chickens are phylogenetically distant vertebrates from 70 humans, but they were instrumental in the field of developmental biology due to the easy access to 71 the embryonated egg. They are used to study neurological and cardiovascular functions (4-6) and 72 provided key findings in B cell development and graft versus host responses (7-9). 73 Modelling human diseases in animals helps elucidating disease pathways and enables the 74 development of new therapies. Although mice are an intensively studied vertebrate model (10), 75 they are often not optimal for modelling particular human diseases. For example, mouse models 76 for familiar adenomatous polyposis (FAP) poo...