High-throughput discovery of essential noncoding sequences for erythropoiesis

项目总结 超过98%的人类基因组由非编码序列组成。这些方面的重要性一直是 由全基因组关联研究(GWAS)强调，该研究已经确定了数千个共同的 与人类特征和疾病易感性相关的遗传变异，其中绝大多数定位于 非编码基因组。此外，随着全基因组测序成本的大幅下降，临床 基因组已经大量繁殖。实现精准医疗潜力和资本化的主要瓶颈 关于GWAS提供的知识，是无法理解和预测 非编码基因组的扰动。直到最近，对非编码基因组的研究还很有限。 到异位异源记者分析、相关生化研究或繁琐的基因敲除实验 模型生物。基因组编辑的进步使人类非编码序列的便捷中断成为可能 在染色质的细胞环境中。最近我们开发了一种技术，Cas9介导的原位饱和 突变，它允许高通量和高分辨率的非编码序列的扰动。我们 假设只有在适当的染色质和细胞环境中进行扰动才能 建立对非编码序列的要求。在这份提案中，我们描述了综合研究，以 将红细胞生成所需的基本非编码序列表征为自然发生的特征- 相关的遗传变异。红细胞生成是一种特别适合研究非编码遗传的系统。 决定因素考虑到其主要的细胞固有性质，直接的临床相关性，以及高可获得性 高质量的人类基因数据、广泛的染色质图谱和可靠的组织培养模型。通过这些研究， 我们将干扰与特征相关的增强子以及非增强子非编码元素，以揭示最小 红细胞生成所需的关键序列。我们将介绍几项技术进步，包括 利用替代核酸酶进行混合筛选、单倍型感知的指导RNA设计、ON-1的预测。 靶标效率和脱靶潜力，以及核酸酶靶标深度测序，以接近核苷酸 关键序列的分辨率确定。我们将利用生物信息学、生物化学和基因组编辑 方法确定与基本顺式作用序列相互作用的关键反式作用因子。总目标 将通过迭代实验测试和改进非编码序列函数模型 分析精化。这些研究旨在对血细胞发育有更好的了解， 寻找治疗血液疾病的新的合理靶点，阐明基因调控的基本机制 和性状遗传力。

PROJECT SUMMARY More than 98% of the human genome consists of noncoding sequences. The importance of these has been emphasized by genome-wide association studies (GWAS), which have identified many thousands of common genetic variants associated with human traits and disease susceptibility, the vast majority of which localize to the noncoding genome. In addition, as the cost of whole genome sequencing has dropped dramatically, clinical genomes have proliferated. A major bottleneck in realizing the potential of precision medicine and capitalizing on knowledge afforded by GWAS is the inability to understand and predict the functional consequences of perturbation of the noncoding genome. Up until recently, studies of the noncoding genome have been limited to ectopic heterologous reporter assays, correlative biochemical studies, or laborious knockout experiments in model organisms. Advances in genome editing have enabled facile disruption of human noncoding sequences in chromatinized cellular contexts. Recently we have developed a technique, Cas9-mediated in situ saturating mutagenesis, which allows the high-throughput and high-resolution perturbation of noncoding sequences. We hypothesize that only by perturbation in the appropriate chromatin and cellular environment can the requirement of noncoding sequences be established. In this proposal we describe comprehensive studies to characterize essential noncoding sequences required for erythropoiesis as marked by naturally occurring trait- associated genetic variation. Erythropoiesis is a particularly apt system to investigate noncoding genetic determinants given its predominantly cell-intrinsic nature, direct clinical relevance, and the availability of high- quality human genetic data, extensive chromatin maps, and faithful tissue culture models. With these studies, we will perturb trait-associated enhancers as well as non-enhancer noncoding elements to reveal minimal critical sequences required for erythropoiesis. We will introduce several technical advances, including utilization of alternative nucleases for pooled screening, haplotype-aware guide RNA design, predictions of on- target efficiency and off-target potential, and nuclease target deep sequencing, to approach nucleotide resolution determination of critical sequences. We will utilize bioinformatic, biochemical, and genome editing methods to define key trans-acting factors interacting with the essential cis-acting sequences. The overall goal will be to develop improved models of noncoding sequence function by iterative experimental testing and analytic refinement. These studies are intended to yield an improved understanding of blood cell development, identify novel rational targets for blood disorders, and illuminate fundamental mechanisms of gene regulation and trait heritability.