Causal Transcriptional Consequences of Human Genetic Variation

描述（由申请人提供）：人类遗传变异转录后果中心（CTCHGV）将开发创新和强大的基因工程方法，并利用它们来识别因果控制基因转录水平的遗传变异。全基因组关联研究（GWAS）发现了许多与疾病和其他表型相关的变异，但实际上可能导致这些疾病的变异很难识别，因为同一单倍型块的附近变异在人群中始终与它们共同发生，因此无法区分特定的致病变异。大约95%的GWAS变异不在基因编码区，其中许多可能与基因表达水平的改变有关。CTCHGV将识别直接控制基因表达的变异，方法是对基因调控区域的变化进行精确的工程组合，从而分解单倍型块，从而使每种变异对基因表达的影响独立于其他变异而被识别出来。为了进行这项分析，CTCHGV将从人类细胞样本中提取~100kbps的基因调控区域，在大肠杆菌中产生精确的变异，并使用锌指核酸酶（ZFNs）有效地诱导重组，将改变的区域重新引入人类细胞。CTCHGV将针对1000个基因进行分析（目标1），并将使用人类诱导多能干细胞（iPS）来研究不同人类细胞类型变异的影响（目标2）。为了探索复杂人体组织中变异的影响，CTCHGV将开发在许多单细胞中转录组水平上测量基因表达的方法，包括结构化组织中的原位基因表达（目的3）。最后，CTCHGV将开发新的先进技术，整合DNA测序和合成，从寡核苷酸构建数千个大型DNA构建体，从而实现非常精确的靶向和高效的zfn性能，并使细胞能够根据形态学以及荧光和标记进行分类（Aim 4）。CTCHGV还将开发人类细胞的直接寡聚介导工程，并创建“标记等位基因”iPS，这将使许多细胞类型中许多对基因等位基因的完整外显子分布易于确定。

DESCRIPTION (provided by applicant): The Center for Transcriptional Consequences of Human Genetic Variation (CTCHGV) will develop innovative and powerful genetic engineering methods and use them to identify genetic variations that causally control gene transcription levels. Genome Wide Association Studies (GWAS) find many variations associated with disease and other phenotypes, but the variations that may actually cause these conditions are hard to identify because nearby variations in the same haplotype blocks consistently co-occur with them in human populations, so that specifically causative ones cannot be distinguished. About 95% of GWAS variations are not in gene coding regions, and many of these presumably associate with altered gene expression levels. CTCHGV will identify the variations that directly control gene expression by engineering precise combinations of changes to gene regulatory regions that break down the haplotype blocks, allowing each variations' effect on gene expression to be discerned independently of the others. To perform this analysis, CTCHGV will extract ~100kbps gene regulatory regions from human cell samples, create precise variations in them in E. coli, and re-introduce the altered regions back into human cells, using zinc finger nucleases (ZFNs) to efficiently induce recombination. CTCHGV will target 1000 genes for this analysis (Aim 1), and will use human induced Pluripotent Stem cells (iPS) to study the effects of variations in diverse human cell types (Aim 2). To explore the effects of variations in complex human tissues, CTCHGV will develop methods of measuring gene expression at transcriptome-wide levels in many single cells, including in situ in structured tissues (Aim 3). Finally, CTCHGV will develop novel advanced technologies that integrate DNA sequencing and synthesis to construct thousands of large DNA constructs from oligonucleotides, that enable very precise targeting and highly efficient performance of ZFNs, and that enable cells to be sorted on the basis of morphology as well as fluorescence and labeling (Aim 4). CTCHGV will also develop direct oligo-mediated engineering of human cells, and create "marked allele" iPS that will enable easy ascertainment of complete exon distributions for many pairs of gene alleles in many cell types. RELEVANCE: CTCHGV methods will yield precise knowledge of effects of human genetic variations on gene expression that will both refine and go beyond GWAS-derived associations between non-coding variations and disease. Powerful new CTCHGV genetic engineering methods will directly enable gene therapy. CTCHGV iPS and single-cell transcriptome technologies will increase understanding of diverse and complex human tissues.