Elucidating the cis-regulatory grammar of human photoreceptors

阐明人类光感受器的顺式调节语法

基本信息

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
项目类别：
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

项目摘要

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的主要尚未解决的挑战之一是了解单个顺序变异会导致疾病。编码变化越来越充分理解，但是我们对非编码变化的影响仍然是基本的。该建议的目的是为分析人类残留疾病中的非编码顺式调节变异。我们假设该序列光感受器特异性顺式调节元件（CRES;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残留疾病中的作用。目前，评估属于非编码DNA内的变体的影响是一个挑战问题，部分原因是我们的能力以高通量方式测定CRE是有限的。为了应对这一挑战，我们已经开发了技术称为CRE-SEQ（通过测序进行顺式调节元件分析）。在Cre-seq中，单个Cres是融合到报告基因，每个基因都包含独特的DNA条形码。由此产生的Cre-Reporter库，包括将数千种结构引入活性视网膜中，并通过报告基因表达来量化用RNA-Seq计数条形码转录本。 Cre-seq有望改变我们测量的能力人类顺式调节变体的影响。为了实现这一目标，我们提出了三个具体目标。在AIM 1中，我们将使用ATAC-SEQ来识别发育中和成熟的人类光感受器中的候选CRE。在AIM 2中，我们将利用计算方法和实验方法（包括CRE-SEQ分析）的组合分析AIM 1中确定的CRE，从而阐明人类的顺式调节语法感受器。 Cre-Seq将在小鼠视网膜和ES细胞衍生的人视网膜中进行器官。这些研究将提供人类光感受器“顺式调节体”的第一个全面观点并将开始破译人类光感受器的顺式调节法。在AIM 3中，我们将使用 “诱变和筛选”的方法来设计一个紧凑的库（150 bp），高度活跃的增强剂针对基因治疗应用中的人类感光体。如果成功，这些研究将建立用于分析非编码顺式调节变化的定量平台，从而使综合在残留疾病背景下全基因组序列数据的解释。此外，他们将设计人类永久基因疗法的新增强子套件。