Utilization of the CRISPR/Cas9 systems to protect chickens against Mareks disease virus and as a tool to dissect virus pathogenesis

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The chicken is an excellent model in developmental biology as well as many other research areas. Beyond that, they are the most important source of animal protein for humans and secures the livelihood of millions of people. A plethora of pathogens threaten the welfare and life of billions of chickens. One of these pathogens is Mareks disease virus (MDV) that causes immunosuppression and deadly lymphomas in susceptible chickens. Despite vaccination that protects against the deadly symptoms, MDV can circulate in vaccinated flocks and continuously develops toward a higher virulence. In the past, this allowed the virus to overcome the vaccine protection, increasing the need for more potent vaccines or other interventions.Bacteria use the flexible CRISPR/Cas9 system to defend themselves from invading viruses, which is now widely used for gene editing. Therefore, we hypothesize that this CRISPR/Cas9 system can be used to protect chickens from deadly pathogens such as MDV and that this system will also help us to investigate cellular factors involved in MDV-induced tumor development. We will address the hypothesis in two specific aims by i) transferring the bacterial immune system CRISPR/Cas9 into chickens to protect them from a deadly MDV challenge and ii) to use CRISPR/Cas9 to characterize the role of cellular CD30 in the development of MDV-induced lymphomas. To achieve aim 1, we will generate Cas9 nuclease expressing chickens as a basic tool for avian research. To obtain chickens with a functional CRISPR/Cas9 targeting MDV, we will mate Cas9 birds with chickens expressing a validated MDV specific gRNA array. Infection of these chickens with MDV will determine if this bacterial immune system can protect the animals from a deadly challenge and if it can prevent spread of the virus. To address aim 2, we will investigate the importance of the cellular surface molecule CD30, which is highly expressed in MDV-induced tumors as well as various lymphomas in humans. We will generate recombinant MDV that expresses an inducible CRISPR/Cas9 system and gRNAs to facilitate abrogation of CD30 gene expression in MDV infected cells at defined time points. Animal experiments will allow us to analyses the effect of CD30 abrogation in the natural target cells and the role of CD30 in tumor formation. Taken together, this project will reveal if the CRISPR/Cas9 system can be used to protect chickens against deadly pathogens and will provide a platform for the analysis of cellular factors such as CD30 in this natural virus/host model for virus-induced tumor formation.