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.

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Background and Aims Crohn’s Disease (CD) is a major subtype of inflammatory bowel diseases (IBD) with increasing incidence and prevalence. Results of studies using available small and large animal models are often poorly translatable to patients, and few CD models show small intestinal pathology. Due to its similarities to humans, the swine has emerged as a highly suitable translational disease model, particularly for testing novel nutritional and technological interventions. Our goal was to develop a physiologically relevant porcine CD model to facilitate translation of findings and interventions towards the clinic. Methods We generated pigs bearing a 93 bp deletion of the adenosine-uracil-rich element (ARE) and a constitutive-decay element within the 3’UTR of the TNF gene. Comparative analysis of physiological, molecular, histological and microbial characteristics was performed between wild-type, TNF ΔARE/+ and TNF ΔARE/ΔARE animals. Alterations in the microbiome were compared to the TNFΔARE mouse model and IBD patients. Results TNF ΔARE pigs recapitulate major characteristics of human CD, including ulcerative transmural ileocolitis, increased abundance of proinflammatory cytokines, immune cell infiltration, and dysbiotic microbial communities. 16s rRNA gene amplicon sequencing revealed enrichment in members belonging to Megasphaera, Campylobacter, Desulfovibrio, Alistipes, and Lachnoclostridum in faecal or mucosa-associated bacteria compared to wild-type littermates. PCA-clustering with a subset of TNFΔARE/+ mice and human IBD patients suggests microbial similarity based on disease severity. Conclusions We demonstrate that the TNFΔARE pig resembles a CD-like ileocolitis pathophenotype recapitulating human disease. The ability to conduct long-term studies and test novel surgical procedures and dietary interventions in a physiologically relevant model will benefit future translational IBD research studies.

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...

Crohns Disease (CD) is incurable, and represents a lifelong burden for patients and its incidence is increasing worldwide. A key contributing factor is a dysregulated immune response. Here we report the generation of genome edited pigs with a deletion of the transcript-destabilizing AU-rich element (ARE) and a constitutive decay element (CDE) in the TNF gene which recapitulate major characteristics of human CD, including ulcerative transmural ileocolitis, increased abundance of proinflammatory cytokines, evidence for impaired integrity of the intestinal epithelial cell barrier, immune cell infiltration, and dysbiotic microbial communities. This physiologically relevant CD model enables human-scale and long-term studies to assess diagnostic, nutritional or microbial interventions, filling the gap for translating findings into the clinic.