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.

Genetically modified animals continue to provide important insights into the molecular basis of health and disease. Research has focused mostly on genetically modified mice, although other species like pigs resemble the human physiology more closely. 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 the generation of transgenic chickens and pigs that constitutively express Cas9 in all organs. These animals are healthy and fertile. Functionality of Cas9 was confirmed in both species for a number of different target genes, for a variety of cell types and in vivo by targeted gene disruption in lymphocytes and the developing brain, and by precise excision of a 12.7-kb DNA fragment in the heart. The Cas9 transgenic animals will provide a powerful resource for in vivo genome editing for both agricultural and translational biomedical research, and will facilitate reverse genetics as well as cross-species comparisons.

B cells have first been described in chickens as antibody producing cells and were named after the Bursa of Fabricius, a unique organ supporting their development. Understanding different factors mediating the early migration of B cells into the bursa of Fabricius is crucial for the study of B cell biology. While CXCL12 (stromal derived factor 1) was found to play an important role in B lymphocyte trafficking in mammals, its role in the chicken is still unknown. Previous studies indicated that chicken CXCL12 and its receptor CXCR4 are simultaneously expressed during bursal development. In this study, we investigated whether the CXCR4/CXCL12 interaction mediates B cell migration in chicken embryo. We used the CRISPR/Cas9 system to induce a CXCR4 knockout in chicken B cells which led to chemotaxis inhibition toward CXCL12. This was confirmed by adoptive cell transfer and inhibition of the CXCR4/CXCL12 interaction by blocking with the small inhibitor AMD3100. In addition, we found that the chicken exhibits similarities to mice when it comes to CXCR4 being dependent on B cell receptor expression. B cells lacking the B cell receptor failed to migrate toward CXCL12 and showed no response upon CXCL12 stimulation. Overall, we demonstrated the significance of CXCR4/CXCL12 in chicken B cell development in vivo and the importance of the B cell receptor in CXCR4 dependent signaling.

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...

The availability of genetically modified mice has facilitated the study of mammalian T cells. No model has yet been developed to study these cells in chicken, an important livestock species with a high availability of gd T cells. To investigate the role of gd and ab T cell populations in birds, we generated chickens lacking these T cell populations. This was achieved by genomic deletion of the constant region of the T cell receptor g or b chain, leading to a complete loss of either gd or ab T cells. Our results show that a deletion of ab T cells but not gd T cells resulted in a severe phenotype in knockout chickens. The ab T cell knockout chickens exhibited granulomas associated with inflammation of the spleen and the proventriculus. Immunophenotyping of ab T cell knockout chickens revealed a significant increase in monocytes and the absence of CD4+ T cells and FoxP3+ regulatory T cells compared to wild type chickens. In addition, we observed a significant decrease in immunoglobulins, B lymphocytes, and changes in the bursa morphology. Our data reveal the consequences of T cell knockouts in chickens and provide new insights into their function in vertebrates.