Comprehensive characterization of variants underlying heart and blood diseases with CRISPR base editing

通过 CRISPR 碱基编辑全面表征心脏和血液疾病的变异

基本信息

批准号：
10627940
负责人：
Daniel Evan Bauer
金额：
$ 192.88万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2026-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10627940
关键词：
Address Biological Biological Assay Blood Blood Cells Blood Platelets Blood Pressure CRISPR interference CRISPR screen CRISPR-mediated transcriptional activation CRISPR/Cas technology Cardiovascular Diseases Cardiovascular system Catalogs Cell Line Cell physiology Cells Cellular Assay Chromatin Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Communities Computing Methodologies Coronary Arteriosclerosis Data Data Collection Data Set Development Disease Disease susceptibility Dissection Dyslipidemias Elements Epigenetic Process Erythrocytes Ethnic Origin Etiology Fetal Hemoglobin Frequencies Gene Expression Gene Frequency Genes Genetic Genetic Variation Genome Genotype Goals Heart Diseases Hematological Disease Hematology Hemoglobin concentration result High Density Lipoprotein Cholesterol Human Human Genetics Hypertension Individual LDL Cholesterol Lipoproteins Leukocytes Link Machine Learning Maps Measures Methods Modeling Molecular Patients Phenotype Positioning Attribute Probability Regulatory Element Reporting Research Risk Risk Factors Scheme Serum Technology Testing Validation Variant Visualization Writing base editing base editor blood lipid causal variant computational pipelines data sharing design disorder risk follow-up functional genomics genetic association genetic variant genome editing genome wide association study genomic variation high throughput screening human disease multi-ethnic multimodality neutrophil next generation novel therapeutic intervention open source precise genome editing predictive modeling programs screening sex single-cell RNA sequencing trait working group

项目摘要

Project Summary How genomic variation influences cellular function is a fundamental problem with tremendous importance for human disease. While it has traditionally been difficult to study the effects of specific sequence variants in an experimentally controlled manner, precise genome editing technologies such as CRISPR base editing enable “writing” of trait-associated variants to cells to unravel their function. In this proposal, we will perform multi-modal genome editing-based functional characterization of a total of 72,000 genomic variants associated with cardiovascular diseases (CVDs) and hematological traits. CVD and blood traits are uniquely suited to functional dissection because cardiovascular (coronary artery disease, high blood pressure, dyslipidemia) and blood traits have among the best-powered multi-ethnic GWAS of any traits, and a substantial component of trait variability can be captured in cellular assays that can be scaled to perform high-throughput screening. We have assembled an interdisciplinary team of world-class experts to provide a generalizable pipeline to unravel the functional impact of CVD and blood trait variants by integrating: (1) rich and ancestry-diverse human genetic discoveries, (2) broadly targetable CRISPR base editors and efficient delivery to primary human cells, (3) high-content assays to profile phenotypes at the levels of chromatin, gene expression and cellular function, and (4) computational methods to design, interpret, visualize, and share experimental results. In Aim 1, we will employ a robust, three-tiered variant prioritization scheme that incorporates evidence for disease association from large, multi-ethnic GWAS as well as probability of causality to nominate variants for functional assessment. Through this scheme, we will select variants associated with red blood cell and neutrophil traits, coronary artery disease, blood pressure, and HDL and LDL cholesterol that span a range of allelic frequencies and likely causality to test in high-throughput cellular assays. In Aim 2, we will perform systematic cellular phenotype-based screens using base editors to install candidate variants as well as CRISPR epigenetic inhibition and activation to explore variant-containing regulatory elements. We will use eight established, scalable cellular phenotypic readouts, each of which will enable us to assess which of 12,000 variants and variant-centered elements alter CVD and blood trait-associated cellular phenotypes. We will additionally employ a high-throughput, genome-integrated chromatin accessibility assay to assess which variants alter chromatin accessibility in trait-relevant cell lines. We will follow up with targeted single cell RNA-seq of 5,600 variants in primary cells from donors of different sex and ethnicity. In Aim 3, we will produce a catalog of validated variants and their association with phenotypes for each of the proposed screens. We will collaborate with other IGVF groups to utilize these data to optimize models that predict functional variants, regulatory elements and disease-causing biological mechanisms, ultimately leading to more complete understanding of the genetic underpinnings of cardiovascular and blood disease risk.

项目概要基因组变异如何影响细胞功能是一个非常重要的基本问题虽然传统上很难研究特定序列变异对人类疾病的影响。通过实验控制的方式，精确的基因组编辑技术（例如CRISPR碱基编辑）使得将与性状相关的变异“写入”细胞以揭示其功能在本提案中，我们将执行多模式。基于基因组编辑的总共 72,000 个基因组变异的功能表征心血管疾病 (CVD) 和血液学特征特别适合功能性。由于心血管（冠状动脉疾病、高血压、血脂异常）和血液特征而进行解剖具有所有性状中最强大的多种族 GWAS 之一，并且具有性状变异性的重要组成部分可以在细胞测定中捕获，并可以扩展以进行高通量筛选。我们组建了一支由世界级专家组成的跨学科团队，为通过整合以下因素来揭示 CVD 和血液性状变异的功能影响：(1) 丰富且血统多样化的人类遗传发现，(2) 广泛靶向的 CRISPR 碱基编辑器和有效递送至原代人类细胞， (3) 在染色质、基因表达和细胞功能水平上分析表型的高内涵分析， (4) 设计、解释、可视化和共享实验结果的计算方法。在目标 1 中，我们将采用稳健的三层变体优先级方案，其中包含疾病证据来自大型、多种族 GWAS 的关联以及提名功能变异的因果关系概率通过这个方案，我们将选择与红细胞和中性粒细胞特征相关的变异，冠状动脉疾病、血压以及跨越一系列等位基因频率的 HDL 和 LDL 胆固醇以及在高通量细胞测定中测试的可能因果关系。在目标 2 中，我们将使用碱基编辑器进行基于细胞表型的系统筛选，以安装候选药物变体以及 CRISPR 表观遗传抑制和激活，以探索包含变体的调控元件。我们将使用八个已建立的、可扩展的细胞表型读数，每个读数都将使我们能够评估 12,000 个变异和以变异为中心的元素中的哪一个会改变 CVD 和血液性状相关的细胞我们还将采用高通量、基因组整合的染色质可及性测定来检测表型。评估哪些变异会改变性状相关细胞系中的染色质可及性，我们将跟进目标单个细胞。对来自不同性别和种族供体的原代细胞中的 5,600 个变异进行细胞 RNA 测序。在目标 3 中，我们将生成经过验证的变体目录及其与每个基因表型的关联我们将与其他 IGVF 小组合作，利用这些数据来优化预测模型。功能变异、调控元件和致病生物机制，最终导致更多完全了解心血管和血液疾病风险的遗传基础。

项目成果

期刊论文数量（1）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Multimodal CRISPR perturbations of GWAS loci associated with coronary artery disease in vascular endothelial cells.

血管内皮细胞中与冠状动脉疾病相关的 GWAS 位点的多模式 CRISPR 扰动。

DOI：
发表时间：
2023-03
期刊：
影响因子：
4.5
作者：
Wünnemann, Florian;Fotsing Tadjo, Thierry;Beaudoin, Mélissa;Lalonde, Simon;Lo, Ken Sin;Kleinstiver, Benjamin P;Lettre, Guillaume
通讯作者：
Lettre, Guillaume