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。改良后的牛可与核移植/克隆技术一起用于培育对牛海绵状脑病（BSE或“疯牛病”）具有抗性的牛。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。第一阶段（概述如下）和最终的第二阶段建议的成功将立即产生医疗影响，减少与食用感染疯牛病的牛肉和牛副产品相关的潜在人类健康风险，加强消费者对牛肉产品安全的信心，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，最终将抗疯牛病的牛引入牛肉和乳制品行业并进行繁殖，也将给医疗领域带来重大好处，包括严重依赖牛副产品的药品和化妆品行业。同样，抗疯牛病的牛将减少目前为确保这些产品不患疯牛病而采用的广泛监测计划和监管措施的需要。去年，欧洲在疯牛病监测上花费了5亿多美元。我们这项建议的具体目标如下：

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.