Development of tools for site-directed analysis of gene function

此应用程序的目标是应用工具和改进方法来 在斑马鱼中进行基因组工程，允许产生诱变和条件性 对基因表达提供空间和时间控制的等位基因。通过这样做，我们将能够 定义有助于开发和实施方法学的基因，以改进我们的 了解可以促进或恢复健康的基因操作。让我们有能力 斑马鱼和其他模型中的特定部位的、未标记的突变等位基因已有很大进展 通过包括TALENS和CRISPR/CAS9系统的定制核酸酶。这些系统运行 基于同样的原理：它们被设计成与基因组中的特定序列结合，并 创造一个双链断裂。在第一个授予周期中，我们利用这一能力 发展可靠的针对短片段的定点基因整合方法 同源序列。在我们的续订应用程序中，我们利用工具、矢量和 为斑马鱼创造可逆等位基因和Cre/Cre-ER系的方法 目标1中的社区。在目标2中，我们将研究改进特定站点的方法 CRISPR/Cas9酶切位点的微同源整合和作用及呈现的新途径 与基因组切点具有微同源性的载体。在目标3中，我们将继续发展和 通过在两个地点举办研讨会和现场访问来实施有针对性的集成资源 梅奥诊所和爱荷华州立大学。制作的工具、技术和斑马鱼线条将 对提供精确的基因编辑技术和关键线路具有直接影响 检查有可能恢复人类健康的基因。我们期待着这些方法 将提高基因编辑的效率，并可很容易地适用于其他模型 有机体和大型动物。在我们看来，这将对建模具有重要的影响 通过利用条件等位基因的能力在动物系统中引起的人类疾病。这些方法， 货物和斑马鱼品系的设计目的是显著增强对 通过利用基因组修饰产生有条件的和可逆转的产品来促进健康 等位基因和反映人类突变的等位基因。

The goals of this application are to apply tools and refine methodologies for genome engineering in zebrafish that allow the creation of mutagenic and conditional alleles that provide spatial and temporal control of gene expression. In doing so we will be able to define genes that aid in the development and implementation of methodology to improve our understanding of genetic manipulations that can promote or restore health. 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. During the first granting cycle, we have leveraged this ability to develop reliable methods for site-specific gene integration directed by short regions of homologous sequence. In our renewal application, we utilize the tools, vectors and methodologies generated to create both revertible alleles and Cre/Cre-ER lines for the zebrafish community in Aim 1. In Aim 2, we will examine methodologies to improve site-specific integration and the role of microhomology at CRISPR/Cas9 cut sites and new ways to present the vector with microhomology to the genomic cut site. In Aim 3, we will continue to develop and implement targeted integration resources by hosting workshops and onsite visits at both the Mayo Clinic and Iowa State University. The tools, techniques and zebrafish lines produced will have direct implications for providing precise gene editing techniques and critical lines to examine genes with the potential to restore human health. We anticipate these methodologies will enhance the efficiency of gene editing and 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 in animal systems through the ability to utilize conditional alleles. The methods, cargos and zebrafish lines are designed to significantly enhance identification of genes that promote health through leveraging genome modification to produce conditional and revertible alleles and alleles that mirror mutations in humans.