Using genomic perturbations to understand trait-associated human genetic variation

项目摘要/摘要 全基因组关联研究(GWAS)已经确定了数千个与 代谢、心血管、自身免疫等疾病。这些变种有可能揭示 支持人类疾病的分子机制，但对它们的解释极具挑战性，因为 大多数都在功能未知的非编码基因组区域内。拟议研究的长期目标 是通过收集全面的调控目录来阐明复杂疾病的分子基础 并阐明非编码遗传变异如何影响基因调控。这项提议将对 高通量基因组扰动和计算分析发现调控的力量 序列，解释非编码的遗传变异，并将疾病相关变异与它们的基因联系起来 监管。研究重点1将利用CRISPR定向系统地发现新的调控序列 平铺删除屏幕，可以发现其他方法看不到的调控序列。这些 筛查将在原代T细胞中进行，并应用于T细胞周围的兆级区域 分化基因，这些基因富含未鉴定的GWAHs。要确定GWAS命中的频率 影响缺乏规范增强子标记的新调控序列，精细映射的GWA变异体将 与屏幕发现的调控序列相交。新的调控序列的功能 将通过缺失来确定，随后进行实验以测量3D染色质接触、基因表达 和细胞增殖。研究焦点2将利用单细胞基因组扰动将数千人 与人类疾病相关的变异，到它们跨多种细胞类型调控的基因。数列 含有潜在原因的GWA变异体将受到CRISPR干扰，基因表达将 用与疾病相关的免疫细胞混合物中的单细胞RNA-SEQ进行测量。使用单元格数据， 受干扰的序列将与特定细胞类型中基因表达的变化有关。预测的变种 对基因表达的调控将通过基因组编辑修改等位基因来验证。预期结果 该项目的主要内容是(I)T细胞分化相关基因调控序列的系统编目，(Ii) 含有GWAHITS和(III)连接的新的无标记调控序列的分子特征 包含GWASHITS的序列和它们在特定细胞类型中调节的基因之间的关系。这项提议将 建立基因组扰动作为解释非编码变体的新策略，发现重要的新 调节生物学，并加速对与疾病相关的变异的机械性理解。

PROJECT SUMMARY/ABSTRACT Genome-wide association studies (GWAS) have identified thousands of genetic variants associated with metabolic, cardiovascular, autoimmune and other diseases. These variants have the potential to reveal molecular mechanisms that underpin human diseases, but their interpretation is extremely challenging, because most are within non-coding genomic regions with unknown function. The long-term goal of the proposed research is to elucidate the molecular basis of complex diseases by assembling comprehensive catalogs of regulatory sequences and illuminating how non-coding genetic variants affect gene regulation. This proposal will leverage the power of high-throughput genomic perturbations and computational analyses to discover regulatory sequences, interpret non-coding genetic variants, and connect disease-associated variants to the genes they regulate. Research Focus 1 will systematically discover novel regulatory sequences using CRISPR-directed tiling deletion screens, which can discover regulatory sequences that are invisible to other approaches. These screens will be performed in primary T cells and applied to megabase-scale regions surrounding T cell differentiation genes, which are rich in uncharacterized GWAS hits. To determine how frequently GWAS hits affect novel regulatory sequences lacking canonical enhancer marks, fine-mapped GWAS variants will be intersected with regulatory sequences discovered by the screens. The function of novel regulatory sequences will be determined with deletions followed by experiments to measure 3D chromatin contacts, gene expression, and cellular proliferation. Research Focus 2 will utilize single-cell genome perturbations to connect thousands of variants associated with human diseases to the genes they regulate across multiple cell types. Sequences containing potentially causal GWAS variants will be targeted with CRISPR interference and gene expression will be measured with single-cell RNA-seq in a mixture of disease-relevant immune cells. Using the single-cell data, perturbed sequences will be connected to changes in gene expression in specific cell types. Variants predicted to regulate gene expression will be validated by modifying alleles with genome editing. The expected outcomes of this project are (i) systematic catalogs of regulatory sequences for genes involved in T cell differentiation, (ii) molecular characterization of novel unmarked regulatory sequences that contain GWAS hits and (iii) connections between sequences containing GWAS hits and genes that they regulate in specific cell types. This proposal will establish genomic perturbations as a new strategy to interpret non-coding variants, uncover important new regulatory biology, and accelerate mechanistic understanding of disease-associated variants.