High-throughput investigation of human genetic variants affecting cholesterol uptake and efflux

项目摘要 胆固醇代谢的遗传差异是导致冠心病(CAD)风险的主要因素， 这是美国最主要的死亡原因。对胆固醇基因的解开继续产生了 治疗心脏病的很有前途的疗法。尽管如此，胆固醇水平的遗传学还远未完全 了解--有几十到数百个基因组区域的变异会有意义地改变胆固醇 然而，我们只能解释这些基因座中一小部分的遗传基础。我们有 建立了一种将CRISPR筛查、基因网络分析和人类生物库相结合的有效方法 编码变量负荷分析以剖析胆固醇摄取和排出的遗传学。通过这条管道，我们 在细胞模型中发现了数十个与低密度脂蛋白(LDL-C)摄取有关的新基因 对于这些编码变体会改变人群中的血清低密度脂蛋白-C水平。在这份提案中，我们将细化和扩展 这条管道对胆固醇的变种、基因和途径有一个连贯的理解 摄取和排出。 在目标1中，我们开创了一种新的管道，将CRISPR筛选、基因网络分析和人类生物库相结合 负荷分析，以表征我们已发现的改变细胞低密度脂蛋白摄取的~500个基因。我们将开发一种 利用大外显子提取稀有编码变异对血清低密度脂蛋白水平影响的灵敏方法 对生物库队列进行排序。我们将通过组合将改变低密度脂蛋白摄取的基因分组到不同的途径中 CRISPR筛查和基因网络分析。我们将对新的候选低密度脂蛋白进行机械性跟进- 在细胞和小鼠体内模型中的C-改变通路。在目标2中，我们将开创一种新的、更敏感的 CRISPR碱基编辑屏幕识别改变肝脏低密度脂蛋白摄取的GWAS相关变体的方法 细胞。然后，我们将使用我们开发的一套计算和实验工具来剖析顺- 这些变异体发挥作用的调节机制，并将它们与控制它们的跨调节输入连接起来。 我们希望将肝细胞摄取低密度脂蛋白的转录驱动因素与它们的顺式调节GWA联系起来。 相关的变异靶点和下游的低密度脂蛋白摄取改变基因，揭示了人类如何 变异会影响血清低密度脂蛋白胆固醇水平。在目标3中，我们将使用CRISPR-Cas9筛查和人类 生物库分析识别与单核/巨噬细胞胆固醇逆转相关的基因和途径 运输，一个被认为在冠心病风险中很重要的过程，但在遗传学上还没有完全理解 水平。我们将剖析涉及胆固醇反向运输的疾病相关遗传机制，帮助 明确巨噬细胞外流在冠心病风险中的作用。 总之，通过高通量CRISPR筛查和随后的机械性后续行动，我们将提供最多 迄今为止，对非编码基因座、基因和通路进行了广泛的实验和计算分析 人类细胞胆固醇摄取和外流的变化。

Project Summary Genetic differences in cholesterol metabolism are major contributors to the risk of coronary artery disease (CAD), which is the leading cause of death in the USA. Unraveling the genetics of cholesterol has continued to yield promising therapeutics for heart disease. Nonetheless, the genetics of cholesterol levels are far from completely understood-- there are dozens to hundreds of genomic regions whose variation meaningfully alters cholesterol levels in the population, yet we can only explain the genetic basis of a small fraction of these loci. We have established a powerful approach combining CRISPR screening, gene network analysis, and human biobank coding variant burden analysis to dissect the genetics of cholesterol uptake and efflux. Through this pipeline, we have identified dozens of new genes that contribute to LDL cholesterol (LDL-C) uptake in cellular models and for which coding variants alter serum LDL-C levels in the population. In this proposal, we will refine and extend this pipeline to develop a coherent understanding of the variants, genes, and pathways underlying cholesterol uptake and efflux. In Aim 1, we pioneer a novel pipeline combining CRISPR screening, gene network analysis, and human biobank burden analysis to characterize ~500 genes we have found to alter cellular LDL-C uptake. We will develop a sensitive approach to extract the effects of rare coding variants on serum LDL-C levels using large exome sequencing biobank cohorts. We will group LDL-C uptake-altering genes into pathways through a combination of CRISPR screening and gene network analysis. We will perform mechanistic follow-up of novel candidate LDL- C-altering pathways in cellular and mouse in vivo models. In Aim 2, we will pioneer a new, more sensitive approach to CRISPR base editing screens to identify GWAS-associated variants that alter LDL-C uptake in liver cells. We will then use a suite of computational and experimental tools we have developed dissect the cis- regulatory mechanisms by which these variants act and connect them to trans-regulatory inputs controlling them. We expect to connect transcriptional drivers of hepatocyte LDL-C uptake with their cis-regulatory GWAS- associated variant targets and downstream LDL-C uptake-altering genes, shedding light on how human genetic variation influences serum LDL-C level. In Aim 3, we will use the pipeline of CRISPR-Cas9 screening and human biobank analysis to identify genes and pathways associated with monocyte/macrophage reverse cholesterol transport, a process thought to be important in CAD risk but which is incompletely understood at the genetic level. We will dissect disease-relevant genetic mechanisms involved in reverse cholesterol transport, helping to define the role of macrophage efflux in CAD risk. In sum, through high-throughput CRISPR screening followed by mechanistic follow-up, we will provide the most extensive experimental and computational analyses to date of the non-coding loci, genes, and pathways that underlie variation in human cellular cholesterol uptake and efflux.