Genome-Editing of the PrP Gene in Bovine Cells

描述(由申请人提供)：我们将证明使用一种新的基因组编辑技术(称为GenEdit)在原代牛成纤维细胞(BF)中修饰牛PrP基因(BoPrP)的可行性。然后，改良的BF可以与核移植/克隆技术一起使用，以产生对牛海绵状脑病(疯牛病)具有抵抗力的牛。基因组编辑技术GenEdit将使用短DNA片段(SDF)在boPrP基因中引入两个核苷酸变化。这一第一阶段(概述如下)和最终第二阶段提案的成功将立即产生医疗影响，减少与食用受疯牛病感染的牛肉和牛副产品相关的潜在人类健康风险，增强消费者对牛肉产品安全性的信心，并避免最终爆发疯牛病对牛肉行业可能造成的灾难性影响。此外，最终将抗疯牛病牛引入牛肉和乳制品行业并进行饲养，也将对医疗领域带来重大好处，包括严重依赖牛副产品的医药和化妆品行业。同样，抵抗疯牛病的牛将减少目前为确保这些产品对疯牛病的安全性而采用的广泛监测计划和监管措施的必要性。去年，欧洲在疯牛病监测上花费了超过5亿美元。我们对这项建议的具体目标如下： 目的1)克隆和鉴定我们希望引入抗性性状的特定品种的boPrP基因序列，并产生SDF分子以引入boPrP基因的无义和错义突变。 目的2)建立一种快速基因编辑检测方法。 目的#3)建立向BF核输送SDF分子的最佳条件。 目的#4)利用基因编辑报告系统LX-MIR建立BF细胞基因编辑事件的频率。 目的#5)在原代BF细胞中使用GenEDIT对boPrP基因进行修饰，并分离出一个细胞克隆，最终用于核移植/克隆，以产生携带该修饰等位基因的牛。

DESCRIPTION (provided by applicant): We will demonstrate the feasibility of using a novel genome editing technology (termed GenEdit) to modify the bovine PrP gene (boPrP) in primary bovine fibroblasts (BF). The modified BFs may then be used with nuclear transfer/cloning technologies to generate cattle resistant to bovine spongiform encephalophy (BSE or "mad cow disease"). The genome editing technology, GenEdit, will be employed using short DNA fragments (SDF) to introduce two nucleotide changes in the boPrP gene. The success of this phase I (outlined below) and the eventual phase II proposal will have an immediate medical impact, reducing the potential human health risk associated with consuming BSE-infected beef and bovine by-products, strengthening the consumer's confidence in the safety of beef products, and circumventing possible catastrophic affects on the beef industry as a consequence of an eventual BSE outbreak. Moreover, the eventual introduction and breeding of BSE-resistant cattle into the beef and dairy industries will also be a major benefit to the medical fields, including drug and cosmetic industries that rely heavily on bovine by-products. Similarly, BSE-resistant cattle will reduce the need for the extensive monitoring programs and regulatory measures currently being employed to assure the safety of these products against BSE. Last year Europe spent over 500 million dollars on BSE monitoring. Our specific aims for this proposal are the following: Aim # 1) clone and characterize the boPrP gene sequence in the specific breeds we wish to introduce the resistant trait, and generate SDF molecules to introduce nonsense and missense mutations in the boPrP gene. Aim # 2) Develop a rapid gene-editing detection assay. Aim # 3) Establish optimal conditions for delivering SDF molecules to BF nuclei. Aim # 4) Establish the frequency on gene editing events in BF cells using a gene-editing reporter system, LX-MIR. Aim # 5) Modify the boPrP gene using GenEdit in primary BF cells and isolate a cell clone for the eventual use in nuclear transfer/cloning for generating cattle carrying this modified allele.