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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：
10601005
负责人：
JOSEPH CORBO
金额：
$ 53.92万
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 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10601005
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 Mutagens 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 blind candidate identification 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未解决的主要挑战之一是了解个体序列如何变异导致疾病。编码变异越来越被理解，但是我们对编码变异的了解非编码变异的影响仍然是基本的。该提案的目标是为人视网膜疾病中非编码顺式调节变异的分析。我们假设这个序列光受体特异性顺式调节元件（克雷斯;例如，增强子/启动子）发挥重要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，评估属于非编码DNA的变异的影响代表了一个具有挑战性的问题，部分原因是我们的能力以高通量方式测定克雷斯是有限的。为了应对这一挑战，我们制定了 CRE-seq（顺式调控元件测序分析）。在CRE-seq中，单独的克雷斯是与报告基因融合，每个基因含有独特的DNA条形码。得到的CRE-报告基因文库，由以下组成：将数千种构建体中的一种引入活视网膜中，并通过以下方法定量报告基因表达：用RNA-seq计数条形码化的转录物。CRE-seq有望彻底改变我们测量人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。目标1：将使用ATAC-seq来鉴定发育和成熟人类光感受器中的候选克雷斯。在目标2中，我们将利用计算和实验方法（包括CRE-seq分析）的组合，分析目标1中鉴定的克雷斯，从而阐明人类的顺式调节语法。光感受器CRE-seq将在小鼠视网膜以及ES细胞衍生的人视网膜细胞中进行。类器官这些研究将提供人类光感受器顺式调节组的第一个全面视图并将开始破译人类光感受器的顺式调节密码。在目标3中，我们将使用 “诱变和筛选”方法来工程化紧凑（150 bp）的高活性增强子文库，在基因治疗应用中靶向人类光感受器。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定量平台，从而实现全面的在视网膜疾病的背景下全基因组序列数据的解释。此外，他们还将设计一个一套用于人视网膜基因治疗新增强子。