Functional characterization of blood cell associated regulatory GWAS hits

描述(由申请人提供)：从全基因组关联研究(GWAS)中出现的绝大多数(&gt；90%)与疾病和性状相关的变异位于基因组的非编码区，目前除了少数缺乏分子机制来解释观察到的与复杂特征的关联。将人类调控DNA目录与Gwas数据叠加，揭示了由DNaseI超敏位点定义的调控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中检查其生物染色质背景下的遗传变异时，我们才能充分了解基因调控和错误调控。故事核酸酶(TALEN)平台可以精确、有针对性地改变细胞的DNA序列。我们已经确定了单TALEN介导的同源重组用于有效敲除单个调控DNA区域的可行性。在R21阶段，我们将在10个单独的调控区域使用工程模板在体内对调控DNA中的候选疾病/性状相关变体进行建模。我们将测试使用K562细胞的TALEN基因组修饰管道的可扩展实施的可行性，并确定显著的操作特征，并展示在原代造血细胞中敲除和插入特定等位基因的可行性和文件效率。我们将测试在编辑站点两侧使用单个或多个TALEN的效率。在R33阶段，我们将扩大TALEN管道过程，以有效地表征GWAS研究中确定的与红细胞表型相关的位置。这些研究将为调控元件的作用提供直接证据，并证明与GWAS相关的SNPs与所观察到的表型有关。

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.