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

鸡是发育生物学以及许多其他研究领域的优秀模型。除此之外，它们是人类最重要的动物蛋白质来源，并保障了数百万人的生计。大量的病原体威胁着数十亿鸡的福利和生命。这些病原体之一是马立克氏病病毒（MDV），导致免疫抑制和致命的淋巴瘤在易感鸡。尽管接种疫苗可以预防致命症状，但MDV可以在接种疫苗的鸡群中传播，并不断发展成更高的毒力。在过去，这使得病毒能够克服疫苗的保护，增加了对更有效的疫苗或其他干预措施的需求。细菌使用灵活的CRISPR/Cas9系统来保护自己免受入侵病毒的侵害，该系统现在被广泛用于基因编辑。因此，我们假设这种CRISPR/Cas9系统可用于保护鸡免受MDV等致命病原体的侵害，并且该系统还将帮助我们研究MDV诱导的肿瘤发展中涉及的细胞因子。我们将通过以下两个具体目标来解决这一假设：i）将细菌免疫系统CRISPR/Cas9转移到鸡中，以保护它们免受致命的MDV攻击; ii）使用CRISPR/Cas9来表征细胞CD 30在MDV诱导的淋巴瘤发展中的作用。为了实现目标1，我们将产生表达Cas9核酸酶的鸡作为禽类研究的基本工具。为了获得具有靶向MDV的功能性CRISPR/Cas9的鸡，我们将Cas9鸟与表达经验证的MDV特异性gRNA阵列的鸡交配。用MDV感染这些鸡将决定这种细菌免疫系统是否可以保护动物免受致命的挑战，以及是否可以防止病毒的传播。为了解决目标2，我们将研究细胞表面分子CD 30的重要性，CD 30在MDV诱导的肿瘤以及人类各种淋巴瘤中高度表达。我们将产生表达诱导型CRISPR/Cas9系统和gRNA的重组MDV，以促进在限定的时间点消除MDV感染的细胞中的CD 30基因表达。动物实验将使我们能够分析CD 30在天然靶细胞中的作用以及CD 30在肿瘤形成中的作用。总之，该项目将揭示CRISPR/Cas9系统是否可用于保护鸡免受致命病原体的侵害，并将为分析这种天然病毒/宿主模型中的细胞因子（如CD 30）提供平台，以用于病毒诱导的肿瘤形成。

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.