Enhancing The Value of Pigs for Agriculture and Biomedical Applications By Using Novel Genome Editing Strategies

项目摘要/摘要 基因工程(GE)动物模型是产生人类疾病生物医学模型的关键 也是为了更好地了解动物生物学。对动物基因组进行有针对性的修改使 动物表现出人类疾病的表型，因此，对于设计和开发新的治疗方法至关重要。 使用GE大型动物模型，如猪，通常会导致临床上相关的结果，因为它们的生理 解剖结构与人类相似。例如，引入导致囊性纤维化的遗传因素和 对猪基因组的免疫缺陷导致转基因猪紧密地总结疾病的症状。然而， 由于建立这样的模型需要大量的时间，通用电气猪在生物医学中并不广泛使用。AS 作为一种大型动物物种，一轮猪的繁育至少需要一年时间，而且往往需要多轮 为了建立GE猪模型，育种是必要的。基因组编辑工具的应用，如CRISPR/CAS 该系统大大提高了对猪基因组进行有针对性的修饰的效果。然而， 对基因组编辑程序和引入所需天数造成的意外基因组更改的担忧 将猪作为大型动物模型进行有针对性的修改，阻碍了该技术的广泛应用。我们的目标是 该项目旨在评估基因组编辑技术的有效性和安全性，并设计新的方法 这将有助于在有针对性的基因组编辑活动后对动物模型进行快速表型鉴定。三个具体目标 是为了达到我们的目标而提出的。首先，我们将生成用于全局检测GE中的偏离目标事件的方法 猪。其次，我们将制定策略，确保基因组编辑过程中的基因组完整性。最后， 我们建议开发一种策略，以快速表型GE胎儿和修改野生型猪的基因组。 第三个目标的目标是与农业和生物医学相关的特征相关的基因。 通过本项目所获得的知识可以应用于扩大转基因猪在生物医学上的应用，同时 对农业生产也有影响。利用基因组预测表型的重要性 为了快速鉴定改良的等位基因和性状，将在这里种植。我们在使用基因组编辑方面的专业知识 将使用技术和GE猪模型来完成拟议的目标。这个项目的成果应该是 通过有效地捕获后续数据，提高GE猪模型在生物医学和农业中的可用性 基因组编辑事件后的表型。我们建议以猪为模型来研究该药的疗效。 提出的策略；然而，我们的发现应该很容易地转移到生产其他动物模型在 还有生物医学和农业上重要的物种。鉴于猪作为动物模型的重要性， 我们的发现应该对美国国立卫生研究院和美国农业部的机构都有利。

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.