Using genomic perturbations to understand trait-associated human genetic variation

利用基因组扰动来了解与性状相关的人类遗传变异

• 批准号: 10292872
• 负责人: GRAHAM MCVICKER
• 金额: $ 57万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-19 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10292872
• 关键词: 3-Dimensional; Affect; Alleles; Autoimmune; Biology; CRISPR interference; Cardiovascular system; Catalogs; Cell Proliferation; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Computer Analysis; Data; Differentiated Gene; Disease; Enhancers; Gene Expression; Gene Expression Regulation; Genes; Genetic Variation; Genome; Genomic Segment; Genomics; Goals; Human Genetics; Immune; Maps; Measures; Metabolic; Molecular; Outcome; Research; T cell differentiation; T-Lymphocyte; Untranslated RNA; Variant; cell type; experimental study; genetic variant; genome editing; genome wide association study; human disease; novel; single-cell RNA sequencing; trait

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.

项目总结/摘要 全基因组关联研究（GWAS）已经确定了数千种与遗传相关的遗传变异。 代谢、心血管、自身免疫和其他疾病。这些变异有可能揭示 这些分子机制是人类疾病的基础，但对其解释极具挑战性，因为 大多数位于功能未知的非编码基因组区域内。拟议研究的长期目标 是阐明复杂疾病的分子基础， 序列，并阐明非编码遗传变异如何影响基因调控。该提案将利用 高通量基因组扰动和计算分析的力量，以发现调控 序列，解释非编码遗传变异，并将疾病相关变异与它们 规范。研究重点1将利用CRISPR指导的系统性发现新的调控序列 平铺删除屏幕，可以发现其他方法不可见的调控序列。这些 筛选将在原代T细胞中进行，并应用于T细胞周围的兆碱基规模区域。 分化基因，其富含未表征的GWAS命中。要确定GWAS命中的频率， 影响缺乏典型增强子标记的新型调控序列，精细定位的GWAS变体将被 与通过筛选发现的调节序列相匹配。新型调控序列的功能 将通过缺失，然后通过实验测量3D染色质接触，基因表达， 和细胞增殖。研究重点2将利用单细胞基因组扰动连接数千个 与人类疾病相关的变异与它们在多种细胞类型中调节的基因有关。序列 含有潜在致病性GWAS变体的基因将被CRISPR干扰靶向，基因表达将 在疾病相关免疫细胞的混合物中用单细胞RNA-seq测量。使用单细胞数据， 受干扰的序列将与特定细胞类型中基因表达的变化相关联。预测变量 将通过基因组编辑修饰等位基因来验证调控基因表达的有效性。预期成果 该项目的主要内容是（i）参与T细胞分化的基因的调控序列的系统目录，（ii） 含有GWAS命中的新的未标记的调节序列的分子表征和（iii）连接 包含GWAS命中的序列和它们在特定细胞类型中调节的基因之间的关系。这项建议会 建立基因组扰动作为解释非编码变异的新策略，发现重要的新 调节生物学，并加速疾病相关变异的机制理解。