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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.

项目概要基因组时代尚未解决的主要挑战之一是了解个体序列如何变异会导致疾病。编码变异越来越被人们所理解，但我们对编码变异的了解非编码变异的影响仍然处于初级阶段。该提案的目标是开发一个平台人类视网膜疾病中非编码顺式调控变异的分析。我们假设该序列光感受器特异性顺式调节元件（CRE；例如增强子/启动子）的变异发挥着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，正在评估非编码 DNA 内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方式测定 CRE 是有限的。为了应对这一挑战，我们开发了称为 CRE-seq（顺式调控元件测序分析）的技术。在 CRE-seq 中，各个 CRE 是与报告基因融合，每个基因都包含独特的 DNA 条形码。由此产生的 CRE 报告库，包括数千个构建体被引入活体视网膜，报告基因表达通过以下方式量化使用 RNA-seq 对带条形码的转录本进行计数。 CRE-seq 有望彻底改变我们衡量人类顺式调节变异的影响。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 来识别发育中和成熟的人类光感受器中的候选 CRE。在目标 2 中，我们将结合计算和实验方法（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控语法光感受器。 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控密码。在目标 3 中，我们将使用 “诱变和筛选”方法设计一个紧凑（150 bp）、高活性增强子文库在基因治疗应用中针对人类光感受器。如果成功的话，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，从而实现全面的分析视网膜疾病背景下全基因组序列数据的解释。此外，他们还将设计一个用于人类视网膜基因治疗的一套新增强剂。