Functional characterization of blood cell associated regulatory GWAS hits

血细胞相关监管 GWAS 命中的功能表征

基本信息

批准号：
8606650
负责人：
JOHN A STAMATOYANNOPOULOS
金额：
$ 18.45万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-03-05 至 2016-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8606650
关键词：
Affect Alleles Biological Blood Cells CD34 gene Cataloging Catalogs Cells Characteristics Chromatin Complex Crohn&apos s disease DNA DNA Sequence Data Disease Distal Employee Strikes Engineering Erythrocytes Erythroid Foundations Functional RNA Gene Expression Regulation Generations Genetic Transcription Genome Genomics Hematopoietic Human Hypersensitivity Individual K562 Cells Knock-out Light Mediating Modeling Modification Molecular Nature Nucleic Acid Regulatory Sequences Phase Phenotype Probability Process RNA Regulation Regulatory Element Report (document)Risk Role Side Site Staging Surveys T-Lymphocyte Techniques Testing Tissues Variant abstracting cell type chromatin modification genetic variant genome wide association study homologous recombination in vivo novel nuclease promoter public health relevance trait transcription factor

项目摘要

DESCRIPTION (provided by applicant): The vast majority (>90%) of disease- and trait-associated variants emerging from genome-wide association studies (GWAS) lie in non-coding regions of the genome, and currently all but a handful lack molecular mechanisms that explain the observed associations with complex traits. Superimposition of the human regulatory DNA catalogue with GWAS data reveals a striking concentration of disease-associated variation precisely within regulatory DNA regions defined by DNaseI hypersensitive sites. We will fully understand gene regulation and mis-regulation only if we are able to examine genetic variants in their biological chromatin context in otherwise isogenic cells. TALE nuclease (TALEN) platforms can alter the DNA sequence of cells in a precise, targeted manner. We have already established the feasibility of single-TALEN- mediated homologous recombination for efficient knockout of individual regulatory DNA regions. In R21 phase, we will model candidate disease/trait-associated variants in regulatory DNA in vivo using engineered templates at 10 individual regulatory regions. We will test the feasibility and determine the salient operating characteristics of a scalable implementation of a TALEN genomic modification pipeline using K562 cells, and demonstrate knockout feasibility and specific allele insertion in primary hematopoietic cells and document efficiencies. We will test the efficiency of a using single or multiple TALENs flanking an editing site. In the R33 phase, we will scale the TALEN pipeline process to efficiently characterize sites identified in GWAS studies as associated with red blood cell phenotypes. These studies will provide a direct proof for the role of regulatory elements and demonstrate the relevance of GWAS associated SNPs as causative for the observed phenotypes.

描述（由申请人提供）：全基因组关联研究 (GWAS) 中出现的绝大多数 (>90%) 疾病和性状相关变异位于基因组的非编码区域，目前除少数外，所有变异都缺乏解释观察到的与复杂特征之间的关联的分子机制。人类调控 DNA 目录与 GWAS 数据的叠加揭示了由 DNaseI 超敏感位点定义的调控 DNA 区域内与疾病相关的变异的惊人集中。只有当我们能够在其他同基因细胞的生物染色质环境中检查遗传变异时，我们才能充分理解基因调控和错误调控。 TALE 核酸酶 (TALEN) 平台可以以精确、有针对性的方式改变细胞的 DNA 序列。我们已经确定了单 TALEN 介导的同源重组有效敲除单个调控 DNA 区域的可行性。在 R21 阶段，我们将使用 10 个单独调控区域的工程模板对体内调控 DNA 中候选疾病/性状相关变异进行建模。我们将测试可行性并确定使用 K562 细胞可扩展实施 TALEN 基因组修饰管道的显着操作特征，并证明原代造血细胞中敲除的可行性和特定等位基因插入并记录效率。我们将测试在编辑位点两侧使用单个或多个 TALEN 的效率。在 R33 阶段，我们将扩展 TALEN 管道流程，以有效地表征 GWAS 研究中确定的与红细胞表型相关的位点。这些研究将为调控元件的作用提供直接证据，并证明 GWAS 相关 SNP 与观察到的表型的因果关系。