Development of tools for site-directed analysis of gene function

这个应用程序的首要目标是创建工具和有效方法来定义 可以促进人类健康的基因。虽然大量的数据已经被编目 关于与复杂人类疾病相关的基因突变和基因表达的变化， 我们对那些可能被增选来恢复患者健康的基因缺乏了解。至 解决这一需求并测试当恢复到野生型功能时促进健康的基因， 我们建议开发突变的、可逆的和条件的等位基因，以提供空间和 基因表达的时间控制。制造特定部位的、未标记的突变等位基因的能力 在斑马鱼和其他模型中，定制的核酸酶已经大大进步，包括 TALENS和CRISPR/CAS9系统。这些系统运行在相同的原则上：它们是 被设计成与基因组中的特定序列结合并产生双链断裂。这个 该计划的目标是利用TALEN和CRISPR/CAS9技术的活动，使 特定部位的双链断裂。这些工具和技术将直接影响到 提供精确的基因编辑技术，以评估基因在疾病中的作用及其 在疾病进展后促进健康的能力。虽然我们将开发这些 斑马鱼的方法学由于其易于基因传递，我们预计会有这些 方法论不仅将提高基因编辑的效率，而且将易于适应 用于其他模式生物和大型动物。在我们看来，这将具有重要的意义 在动物系统中模拟人类疾病和健康的意义 能够使可预测的等位基因、小核苷酸多态与相关基因相似 人类疾病和条件等位基因，以测试基因恢复健康的能力。

The overarching goal of this application is to create tools and efficient methods to define genes that can promote human health. While a tremendous amount of data has been cataloged on gene mutation and changes in gene expression associated with complex human disease, our understanding of those genes that could be co-opted to restore patient health is lacking. To address this need and test for genes that when restored to wild type function promote health, we propose develop mutagenic, revertible and conditional alleles that provide spatial and temporal control of gene expression. The ability to make site-specific, untagged mutant alleles in zebrafish and other models has been greatly advanced by custom nucleases that include TALENs and CRISPR/Cas9 systems. These systems operate on the same principle: they are designed to bind to specific sequences in the genome and create a double strand break. The goals of this proposal leverage the activities of TALEN and CRISPR/Cas9 technologies to make site-specific double strand breaks. These tools and techniques will have direct implications for providing precise gene editing techniques to assess the roles of genes in disease and their ability to promote health following disease progression. While we will develop these methodologies in zebrafish due to their ease of gene delivery, we anticipate these methodologies will not only enhance the efficiency of gene editing but will be readily adaptable for use in other model organisms and large animals. In our opinion, this will have important implications for modeling human disease and health in animal systems by greatly enhancing the ability to make predictable alleles, small nucleotide polymorphisms similar to those associated with human disease, and conditional alleles to test for the ability of a gene to restore health.