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

项目总结/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 与复杂性状和常见疾病相关。大多数变异体都定位在98%的基因组上， 非编码，其靶基因或功能在很大程度上未知。这就是变量到函数问题（V2 F）， 解决它仍然是人类遗传学研究的主要障碍。为了帮助解决V2 F问题，我建议开发模块化的 结合GWAS变体优先排序方法和合并单细胞CRISPR靶向筛选的工作流程 基因鉴定我开发了一种综合方法，将高度多基因的血液性状GWAS和 在人红系祖细胞模型（K562）中进行合并的单细胞CRISPR抑制（CRISPRi）筛选， 鉴定靶基因：用单细胞测序系统靶向和抑制非编码GWAS基因座 （STING-seq）。STING-seq可以以大规模平行的方式功能性地剖析多个GWAS基因座， 顺式和反式调节网络中的靶基因。在这里，我将进一步开发STING-seq并检查其 其他GWAS特征及其细胞模型的可推广性。首先，我将使用精确变体扩展STING-seq 用于GWAS变体高通量测量的插入、显影碱基编辑STING-seq（Bee-STING） 对靶基因和调控网络的影响。其次，我将为GWAS变体开发模块化工作流 STING-seq的优先级，以不同的选择标准靶向变体集，以增加STING-seq的 靶基因和调控网络发现率。第三，我将把STING-seq集中在新的GWAS特征和细胞上， 模型来检验它的普遍性，首先对另一个高度多基因复杂性状，骨， 矿物质密度，与人成骨细胞模型（hFOB）。从长远来看，这些目标将有助于解决V2 F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人类遗传学研究的认识。 GWAS变异体因果地影响复杂性状和常见疾病。我有一个全面的训练计划 与我的主要导师Neville Sanjana博士（基因工程）和Tuuli Lappalainen博士（基因 我的导师委员会成员，大卫诺尔斯博士（机器学习），Aravinda博士 查克拉瓦蒂（人类遗传学）、查尔斯·法伯博士（骨骼生物学）和我的合作者尤金·卡采维奇博士 （统计方法）。这个计划将继续我的培训，在解剖GWAS变体功能与多个 计算和实验方法，沿着额外的培训，在赠款写作，指导学生， 教学课程，并在研究会议上发言。完整的导师委员会将指导我 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 市场纽约基因组中心是我理想的培训地点，因为它有先进的设施， 丰富的职业和智力发展机会，以及合作研究环境。后 完成这个培训计划，我将有能力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。

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.