Linking GWAS variants to function with single-cell pooled CRISPR screens

将 GWAS 变体与单细胞 CRISPR 筛选结合起来发挥作用

基本信息

批准号：
10571493
负责人：
John Allan Morris
金额：
$ 10.56万
依托单位：
NEW YORK GENOME CENTER
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-02-16 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10571493
关键词：
Automobile Driving Biological Assay Biology Blood Bone Density Bone Diseases CRISPR screen Catalogs Cell model Cell surface Cells Clustered Regularly Interspaced Short Palindromic Repeats Code Committee Members Complex Complex Genetic Trait Cytosine Data Development Disease Drug Targeting Educational process of instructing Environment Epigenetic Process Erythroid Progenitor Cells Foundations Gene Expression Gene Expression Regulation Genes Genetic Engineering Genetic Research Genome Genome engineering Grant Haplotypes Hematological Disease Human Human Genetics Interdisciplinary Study K-562 Lead Link Linkage Disequilibrium Literature Location Machine Learning Maps Measurement Mentors Mentorship Methods Modeling Mutation New York Nucleotides Osteoblasts Osteoporosis Phase Phenotype Positioning Attribute Proteins Regulatory Element Research Research Project Grants Selection Criteria Signal Transduction Statistical Methods Testing Training Training Programs Untranslated RNA Variant Writing base editing base editor bone bone cell career causal variant cell type flexibility functional genomics genetic association genetic variant genome wide association study genomic data improved insight job market method development mineralization multimodality network dysfunction new therapeutic target novel programs single cell sequencing student mentoring symposium therapeutic candidate therapeutic target trait

项目摘要

PROJECT SUMMARY/ABSTRACT Genome-wide association studies (GWAS) have identified thousands of common and rare genetic variants associated with complex traits and common diseases. Most variants map to the 98% of the genome that is noncoding, with their target genes or function largely unknown. This is the variant-to-function problem (V2F), and solving it remains a major hurdle in human genetics research. To help solve V2F, I propose to develop modular workflows combining GWAS variant prioritization methods and pooled single-cell CRISPR screens for target gene identification. I have developed an integrative approach combining highly polygenic blood trait GWASs and pooled single-cell CRISPR inhibition (CRISPRi) screens in a human erythroid progenitor cell model (K562), to identify target genes: Systematic Targeting and Inhibition of Noncoding GWAS loci with single-cell sequencing (STING-seq). STING-seq can functionally dissect multiple GWAS loci in a massively parallel fashion, identifying target genes in cis as well as trans-regulatory networks. Here, I will develop STING-seq further and examine its generalizability for other GWAS traits and their cell models. First, I will expand STING-seq with precise variant insertion, developing base editing STING-seq (Bee-STING) for high-throughput measurements of GWAS variant effects on target genes and regulatory networks. Second, I will develop modular workflows for GWAS variant prioritization for STING-seq, targeting sets of variants with distinct selection criteria to increase STING-seq’s target gene and regulatory network discovery rate. Third, I will focus STING-seq on new GWAS traits and cell models to examine its generalizability, first piloting STING-seq for another highly polygenic complex trait, bone mineral density, with a human osteoblast cell model (hFOB). In the long-term, these aims will help solve V2F for human genetics research, as their continued development and application will improve our understanding of how GWAS variants causally influence complex traits and common diseases. I have a comprehensive training plan in place with my primary mentors, Dr. Neville Sanjana (genome engineering) and Dr. Tuuli Lappalainen (gene regulation), my mentorship committee members, Dr. David Knowles (machine learning), Dr. Aravinda Chakravarti (human genetics), Dr. Charles Farber (bone biology), and my collaborator Dr. Eugene Katsevich (statistical methods). This plan will continue my training in dissecting GWAS variant function with multiple computational and experimental approaches, along with additional training in grant writing, mentoring students, teaching courses, and presenting at research conferences. The full mentorship committee will direct me to pertinent literature, offer advice on my research program, and provide guidance as I navigate the academic job market. The New York Genome Center is the ideal training location for me, given its cutting-edge facilities, plentiful opportunities for career and intellectual development, and collaborative research environment. Upon completion of this training program, I will be well-positioned to lead my own interdisciplinary research lab and become a leader in the fields of human complex traits genetics and genome engineering.

项目摘要/摘要全基因组关联研究（GWAS）已经确定了数千种常见和罕见的遗传变异与复杂的特征和常见疾病有关。大多数变体映射到98％的基因组非编码，其目标基因或功能在很大程度上未知。这是变体到功能问题（V2F），并且解决它仍然是人类遗传学研究的重大障碍。为了帮助解决V2F，我建议开发模块化结合GWAS变体优先级方法和目标的单细胞CRISPR屏幕的工作流程基因鉴定。我已经开发了一种结合高度多基因的血液特质gwass和的综合方法人类红细胞祖细胞模型（K562）中的汇总单细胞CRIS抑制（CRISPRI）屏幕识别靶基因：使用单细胞测序的非编码GWAS基因座的系统靶向和抑制（sting-seq）。 Sting-seq可以以大规模平行的方式在功能上剖析多个GWAS基因座，从而识别靶基因以及跨调节网络中的靶基因。在这里，我将进一步开发sting-seq并检查它其他GWAS性状及其细胞模型的概括性。首先，我将使用精确变体扩展sting-seq 插入，开发基础编辑刺激序言（蜜蜂），用于GWAS变体的高通量测量对目标基因和调节网络的影响。其次，我将开发GWAS变体的模块化工作流程 sting-seq的优先级，针对具有不同选择标准的变体集以增加sting-seq 靶基因和监管网络发现率。第三，我将把Sting-Seq集中在新的GWAS特征和细胞上检查其推广性的模型，首先是针对另一个高度多基因复杂性状的刺激性seq 矿物质密度，具有人类成骨细胞模型（HFOB）。从长远来看，这些目标将帮助解决V2F 人类遗传学研究，因为它们的持续发展和应用将提高我们对 GWAS变体因果影响复杂的特征和常见疾病。我有一个全面的培训计划与我的主要导师Neville Sanjana博士（基因组工程）和Tuuli Lappalain博士（Gene）法规），我的导师委员会成员，大卫·诺尔斯（David Knowles）（机器学习），阿拉文达（Aravinda）博士 Chakravarti（人类遗传学），Charles Farber博士（骨骼生物学）和我的合作者Eugene Katsevich博士（统计方法）。该计划将继续我的培训，以剖析多个GWAS变体功能计算和实验方法，以及授予写作，心理学生的其他培训，教学课程，并在研究会议上介绍。完整的巡回委员会将指导我相关文献，在我的研究计划中提供建议，并在我浏览学术工作时提供指导市场。鉴于其尖端设施，纽约基因组中心是我的理想培训地点充足的职业和智力发展和协作研究环境的机会。之上完成该培训计划的完成，我将有良好的位置领导自己的跨学科研究实验室和成为人类复杂性状遗传学和基因组工程领域的领导者。