Elucidating the cis-regulatory grammar of human photoreceptors

阐明人类光感受器的顺式调节语法

• 批准号: 10372052
• 负责人: JOSEPH CORBO
• 金额: $ 52.64万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10372052
• 关键词: ATAC-seq; Address; Adult; Affect; Affinity; Bar Codes; Binding Sites; Bioinformatics; Biological Assay; Blindness; CRISPR/Cas technology; Code; Complex; Cone; DNA; Data; Data Set; Disease; Engineering; Enhancers; Gene Expression; Genes; Genetic; Genetic Variation; Genome; Genomics; Goals; Heritability; Human; Human Genome; Individual; Knowledge; Libraries; Location; Maps; Measures; Mendelian disorder; Modeling; Mus; Mutagenesis; Mutation; Nucleic Acid Regulatory Sequences; Organoids; Photoreceptors; Play; Publishing; Regulatory Element; Reporter; Reporter Genes; Retina; Retinal Diseases; Retinal gene therapy; Rod; Role; Techniques; Testing; Therapeutic; Transcript; Untranslated RNA; Variant; Vertebrate Photoreceptors; Vision; base; blind; causal variant; cell type; embryonic stem cell; epigenomics; falls; gene therapy; genetic analysis; genetic variant; genome wide association study; genome-wide; human disease; improved; novel diagnostics; novel strategies; novel therapeutics; promoter; screening; transcription factor; transcriptome sequencing; whole genome

Project Summary One of the major unsolved challenges of the genomic era is to understand how individual sequence variation contributes to disease. Coding variation is increasingly well understood, but our knowledge of the effects of non-coding variation remains rudimentary. The goal of this proposal is to develop a platform for the analysis of non-coding cis-regulatory variation in human retinal disease. We hypothesize that sequence variants in photoreceptor-specific cis-regulatory elements (CREs; e.g., enhancer/promoters) play an important role in retinal disease by altering the expression levels of disease-related genes. Currently, assessing the effects of variants that fall within non-coding DNA represents a challenging problem, in part, because our ability to assay CREs in a high-throughput fashion is limited. To address this challenge, we have developed a technique called CRE-seq (Cis-Regulatory Element analysis by sequencing). In CRE-seq, individual CREs are fused to reporter genes, each containing a unique DNA barcode. The resultant CRE-reporter library, consisting of thousands of constructs, is introduced into living retina, and reporter gene expression is quantified by counting barcoded transcripts with RNA-seq. CRE-seq promises to revolutionize our ability to measure the effects of human cis-regulatory variants. To achieve this goal, we propose three Specific Aims. In Aim 1, we will use ATAC-seq to identify candidate CREs in both developing and mature human photoreceptors. In Aim 2, we will utilize a combination of computational and experimental approaches (including CRE-seq analysis) to analyze the CREs identified in Aim 1 and thereby elucidate the cis-regulatory grammar of human photoreceptors. CRE-seq will be performed in both mouse retinas as well as ES cell-derived human retinal organoids. These studies will provide the first comprehensive view of the human photoreceptor 'cis-regulome' and will begin to decipher the cis-regulatory code of human photoreceptors. In Aim 3, we will use a ‘mutagenesis and screening’ approach to engineer a library of compact (150 bp), highly active enhancers for targeting human photoreceptors in gene therapy applications. If successful, these studies will establish a quantitative platform for the analysis of non-coding cis-regulatory variation, thereby enabling comprehensive interpretation of whole-genome sequence data in the context of retinal disease. In addition, they will engineer a suite of new enhancers for human retinal gene therapy.

项目摘要 基因组时代尚未解决的主要挑战之一是理解个体序列是如何 变异导致疾病。编码变化越来越被理解，但我们对编码变化的了解 非编码变异的影响仍然很小。这项提议的目标是为 人类视网膜疾病中非编码顺式调控变异的分析。我们假设这一序列 光感受器特异性顺式调节元件(CRE；例如，增强子/启动子)的变体起着重要的作用 通过改变疾病相关基因的表达水平，在视网膜疾病中发挥作用。目前，正在评估 属于非编码DNA的变异的影响是一个具有挑战性的问题，部分原因是我们的能力 以高通量的方式测定Cres是有限的。为了应对这一挑战，我们开发了一种 这项技术被称为CRE-SEQ(顺式--通过测序进行调控元件分析)。在CRE-seq中，单个CRE是 融合到报告基因，每个都包含一个唯一的DNA条形码。得到的Cre-Report文库包括 在数以千计的结构中，被引入活的视网膜，报告基因的表达通过 用RNA-seq对条码转录本进行计数。CRE-SEQ承诺彻底改变我们衡量 人类顺式调控变异体的影响。为了实现这一目标，我们提出了三个具体目标。在目标1中，我们 将使用ATAC-seq在发育中和成熟的人类光感受器中识别候选Cre。在目标2中， 我们将利用计算和实验相结合的方法(包括CRE-SEQ分析)来 分析目标1中确定的CRE，从而阐明人类的顺式调节语法 光感受器。Cre-seq将在小鼠视网膜和胚胎干细胞来源的人视网膜上进行。 有机化合物。这些研究将为人类光感受器的顺式调节体提供第一个全面的视角 并将开始破译人类光感受器的顺式调控密码。在目标3中，我们将使用 设计紧凑(150个碱基)高效增强子文库的“诱变和筛选”方法 基因治疗应用中的靶向人光感受器。如果成功，这些研究将建立一个 用于分析非编码顺式调控变异的量化平台，从而使全面 视网膜疾病背景下全基因组序列数据的解释。此外，他们还将设计一种 一套用于人类视网膜基因治疗的新增强剂。