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.

项目概要/摘要全基因组关联研究 (GWAS) 已识别出数千种常见和罕见的遗传变异大多数与复杂的性状和常见疾病相关。非编码，其目标基因或功能很大程度上未知，这就是变体到功能问题（V2F），并且解决它仍然是人类遗传学研究的一个主要障碍。为了帮助解决 V2F，我建议开发模块化。结合 GWAS 变异优先排序方法和针对靶点的单细胞 CRISPR 筛选的工作流程我开发了一种将高度多基因血液性状 GWAS 与基因鉴定相结合的综合方法。在人红系祖细胞模型 (K562) 中进行混合单细胞 CRISPR 抑制 (CRISPRi) 筛选，以识别靶基因：通过单细胞测序系统定位和抑制非编码 GWAS 位点 (STING-seq)。STING-seq 可以以大规模并行方式对多个 GWAS 位点进行功能分析，识别在这里，我将进一步开发 STING-seq 并检查其。首先，我将使用精确变体扩展 STING-seq。插入、开发碱基编辑 STING-seq (Bee-STING)，用于 GWAS 变体的高通量测量其次，我将为 GWAS 变异开发模块化工作流程。 STING-seq 的优先级，针对具有不同选择标准的变体集，以提高 STING-seq 的效率第三，我将把 STING-seq 重点放在新的 GWAS 性状和细胞上。模型来检查其普遍性，首先针对另一个高度多基因的复杂性状（骨）进行 STING-seq 试点从长远来看，这些目标将有助于解决 V2F 问题。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如何 GWAS 变异会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 Lappalainen 博士（基因工程）一起就位。监管），我的导师委员会成员，David Knowles 博士（机器学习），Aravinda 博士 Chakravarti（人类遗传学）、Charles Farber 博士（骨生物学）和我的合作者 Eugene Katsevich 博士（统计方法）。这个计划将继续我对多个 GWAS 变异功能进行剖析的训练。计算和实验方法，以及拨款写作、指导学生等方面的额外培训，教授课程，并在研究会议上发表演讲，完整的指导委员会将指导我：相关文献，为我的研究计划提供建议，并在我从事学术工作时提供指导纽约基因组中心拥有先进的设施，是我理想的培训地点，充足的职业和智力发展机会以及协作研究环境。完成本培训计划后，我将能够领导我自己的跨学科研究实验室成为人类复杂性状遗传学和基因组工程领域的领导者。