Development of tools for site-directed analysis of gene function

此应用程序的目标是应用工具并改进方法， 斑马鱼的基因组工程，允许创造诱变和条件 提供基因表达的空间和时间控制的等位基因。这样我们就能 定义基因，帮助开发和实施方法，以改善我们的 了解可以促进或恢复健康的基因操作。的能力 在斑马鱼和其他模型中，位点特异性的、未标记的突变等位基因已经有了很大的进展 通过包括TALEN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， 造成双链断裂在第一个授予周期中，我们利用这一能力， 开发可靠的方法，用于由短区域指导的位点特异性基因整合， 同源序列在我们的更新应用程序中，我们利用工具，矢量和 产生用于产生斑马鱼的可回复等位基因和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地点的 CRISPR/Cas9切割位点处的微同源性的作用以及呈现 与基因组切割位点具有微同源性的载体。在目标3中，我们将继续开发和 通过举办研讨会和现场访问， 马约诊所和爱荷华州州立大学。工具，技术和斑马鱼生产线将 对于提供精确的基因编辑技术和关键线路， 检测有可能恢复人类健康的基因。我们预计这些方法 这将提高基因编辑的效率，并将很容易适用于其他模型 生物和大型动物。在我们看来，这将对建模产生重要影响 通过利用条件等位基因的能力在动物系统中引起人类疾病。这些方法， cargos和斑马鱼品系被设计为显著增强对基因的识别， 通过利用基因组修饰来产生有条件的和可逆转的 等位基因和与人类突变相对应的等位基因。

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.