Project Summary/Abstract Genetically engineered (GE) animal models are essential for generating biomedical models for human disease and for gaining a better understanding of animal biology. Targeted modification of the animal genome allows the animals to present human disease phenotypes, and therefore, are critical to design and develop novel treatments. The use of GE large animal models such as pigs often results in clinically relevant outcomes as their physiology and anatomy resemble humans. For example, introducing genetic elements responsible for cystic fibrosis and immunodeficiency to the pig genome induces GE pigs to closely recapitulate symptoms of the diseases. However, GE pigs are not widely available in biomedicine due to the amount of time required to establish such models. As a large animal species, a single round of breeding in pigs takes at least one year and often multiple rounds of breeding is necessary to establish GE pig models. Application of genome editing tools, such as the CRISPR/Cas system, has significantly improved efficacy to introduce targeted modifications to the pig genome. However, concerns over unintended genome alterations from genome editing procedure and days required to introduce targeted modifications in pigs as a large animal model impedes wide use of the technology. Our objective of this project is to evaluate the efficacy and safety of genome editing technology and design novel approaches that will assist in rapid phenotyping of animal models after a targeted genome editing event. Three specific aims are proposed to reach our goal. First, we will generate methods for global detection of off-targeting events in GE pigs. Secondly, we will develop strategies to secure genome integrity during the genome editing process. Finally, we propose to develop a strategy to rapidly phenotype GE fetuses and to modify the genome of wild-type pigs. Targets of this third aim are genes associated with traits that are relevant in both agriculture and biomedicine. The knowledge obtained from this project can be implemented to expand the use of GE pigs in biomedicine while also having an impact on agriculture production. The importance of using the genome to predict the phenotype for rapid identification of improved alleles and traits will be grown here. Our expertise in using genome editing technology and GE pig models will be employed to complete the proposed aims. Outcomes of this project should increase the availability of GE pig models in biomedicine and agriculture by effectively capturing subsequent phenotypes after genome editing events. We propose to utilize pigs as a model to investigate the efficacy of the proposed strategies; however, our findings should be easily transferred to producing other animal models in biomedicine and agriculturally important species, as well. Given the importance of pigs used as animal models, our findings should be beneficial to both NIH and USDA agencies.