Development of tools for site-directed analysis of gene function

基因功能定点分析工具的开发

• 批准号: 9457505
• 负责人: Jeffrey J Essner
• 金额: $ 74万
• 依托单位: IOWA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2020-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9457505
• 关键词: Address; Alleles; Animal Model; Animals; Area; Binding; Biochemical; Bioinformatics; Biological Models; CRISPR/Cas technology; Catalogs; Cells; Collaborations; Communities; Complex; Custom; DNA; DNA Double Strand Break; Data; Development; Disease; Disease Progression; Embryo; Gene Delivery; Gene Expression; Gene Mutation; Gene-Modified; Generations; Genes; Genetic Polymorphism; Genome; Genome engineering; Genomics; Goals; Health; Health Promotion; Human; Label; Laboratories; Machine Learning; Mediating; Methodology; Methods; Microinjections; Modeling; Modification; Mutagenesis; Mutate; Mutation; Nucleotides; Oligonucleotides; Operating System; Optics; Pathway interactions; Patients; Production; Proteins; Publications; Publishing; Reagent; Recording of previous events; Recovery; Regenerative Medicine; Research; Research Personnel; Role; Site; System; Systems Integration; Techniques; Technology; Testing; Tissues; Transgenes; Validation; Work; Zebrafish; cost; design; experimental study; functional genomics; gene function; gene therapy; homologous recombination; human disease; human model; improved; induced pluripotent stem cell; insight; interest; method development; mutant; nuclease; recombinase; repaired; site-specific integration; therapeutic development; therapeutic target; tool; tool development; transcription activator-like effector nucleases; vector

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. First, we propose to develop a suite of vectors to make integration alleles that are highly mutagenic and allow production of conditional and revertible alleles. Second, we propose to develop methods to generate predictable alleles in zebrafish at TALEN- and CRISPR/Cas9-induced double strand break sites by invoking the microhomology mediated end-joining pathway. Third, leveraging our preliminary data, we propose to improve methods for homology directed repair with oligonucleotides to create disease associated alleles in zebrafish and for site-specific integration using homologous recombination at TALENs and CRISPR/Cas9 cut sites. Fourth, we propose use single-strand annealing at TALENs and CRISPR/Cas9 cut sites to promote precise transgene integration to make tagged and highly mutagenic allele. 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 predictible 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.

这个应用程序的首要目标是创建工具和有效的方法来定义 能促进人类健康的基因。虽然大量的数据已经被编目 与复杂人类疾病相关的基因突变和基因表达变化， 我们对那些可以用来恢复病人健康的基因缺乏了解。到 解决这一需要并测试当恢复到野生型功能时促进健康的基因， 我们建议开发诱变的、可回复的和条件性的等位基因， 基因表达的时间控制。制造位点特异性的无标签突变等位基因的能力 在斑马鱼和其他模型中的应用已经通过定制核酸酶得到了极大的改进， TALEN和CRISPR/Cas9系统。这些系统的工作原理相同： 被设计为与基因组中的特定序列结合并产生双链断裂。的 该提案的目标是利用TALEN和CRISPR/Cas9技术的活动， 位点特异性双链断裂。首先，我们建议开发一套载体， 整合等位基因是高度致突变的，并允许产生条件性和可逆性的 等位基因其次，我们建议开发方法，以产生可预测的等位基因在斑马鱼在 TALEN和CRISPR/Cas9通过调用微同源性诱导双链断裂位点 介导的末端连接途径。第三，利用我们的初步数据，我们建议改善 用寡核苷酸进行同源定向修复以产生疾病相关等位基因的方法 并使用TALEN处的同源重组进行位点特异性整合， CRISPR/Cas9切割位点。第四，我们建议在TALEN处使用单链退火， CRISPR/Cas9切割位点，以促进精确的转基因整合， 致突变等位基因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促进 疾病进展后的健康状况。虽然我们将在斑马鱼中开发这些方法， 由于它们易于基因递送，我们预计这些方法不仅将增强 基因编辑的效率，但将容易适用于其他模式生物， 大型动物。在我们看来，这将对人类疾病建模产生重要影响 和健康的动物系统，大大提高了能力，使可预测的等位基因，小 与人类疾病相关的核苷酸多态性相似， 等位基因来测试基因恢复健康的能力。