High-throughput dissection of transcriptional regulation in kidney disease

肾脏疾病转录调控的高通量解析

• 批准号: 10645854
• 负责人: Josh Tycko
• 金额: $ 8.47万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10645854
• 关键词: Affect; Autosomal Dominant Polycystic Kidney; Award; Binding; Biological Assay; C-terminal; CRISPR interference; CRISPR screen; CRISPR/Cas technology; Cell Nucleus; Cell membrane; Cells; Characteristics; Chemicals; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cyst; Cystic kidney; DNA; DNA Binding Domain; DNA-Binding Proteins; Data; Development; Disease; Disease model; Dissection; Elements; Engineering; Enhancers; Exhibits; Fellowship; Gene Dosage; Gene Expression; Gene Silencing; Gene Targeting; Genes; Genetic Transcription; Genome; Genomics; Goals; Guide RNA; Health; Heterochromatin; Histones; Human; Human Genome; Integral Membrane Protein; Kidney Diseases; Kidney Failure; Knowledge; Lead; Learning; Libraries; Light; Link; Maps; Measurement; Measures; Methods; Modification; Mutation; Nature; Organoids; Pathway interactions; Patients; Persons; Phase; Phenotype; Prevalence; Process; Proliferating; Proprotein Convertase 1; Proteins; Proteome; Regulation; Regulatory Element; Reporter; Research; Research Methodology; Resources; Science; Signal Transduction; System; Tail; Techniques; Technology; Tertiary Protein Structure; Testing; Therapeutic; Toxic effect; Training; Transcriptional Regulation; Untranslated RNA; Variant; Work; Writing; advanced disease; career; cell type; chromatin modification; combinatorial; disease phenotype; epigenome; epigenomics; experimental study; gene therapy; genetic regulatory protein; genome-wide; high throughput screening; high throughput technology; improved; innovation; method development; novel; polycystic kidney disease 1 protein; promoter; protein function; rational design; recruit; response; skills; technology development; therapeutic gene; tool

Chromatin modifications are involved in all basic DNA-templated processes in human cells including transcription, and are mis-regulated in many diseases. With CRISPR-targeting techniques, we can write particular chromatin modifications and then measure how gene expression changes in response. This will enable us to understand how gene expression changes in disease states and how to rationally design therapeutics to reverse those changes. One of the challenges to using CRISPR perturbations to study non-coding regulatory elements is CRISPR off- target activity. Here, we show that off-target effects in non-coding perturbation experiments can be associated with significant toxicity in human cells, not only with DNA-cleaving Cas9, but also with epigenome-modifying CRISPRi/a tools. After removing off-target-prone guide RNAs, we can use CRISPR to accurately link non-coding regulatory elements with genes. These perturbation experiments are critical to learn the causal functions of chromatin modifications at specific genomic elements. However, most experiments to date have used CRISPRi, with one particular KRAB domain that establishes one particular heterochromatin state. Existing tools to manipulate chromatin state are largely drawn from a small fraction of the thousands of natural chromatin regulatory complexes; most suffer from partial or transient effects, and exhibit high variability across loci and cell types. A more complete toolbox of compact, efficient domains for pathway-specific chromatin perturbations will transform our ability to determine the causal function of particular chromatin modifications across the human genome. Here, we propose to systematically measure the gene expression effects of recruiting chromatin regulator protein domains to a promoter. This is made possible by our recent development of a high-throughput chromatin regulator recruitment assay in human cells, capable of measuring activity for tens of thousands of regulator domains simultaneously. Using this system, we will recruit-and-release CR variants from the promoter, and then measure the magnitude and permanence of transcriptional silencing at a reporter locus. We will then use epigenomic mapping assays to determine the chromatin modifications that underpin the silencing functions of these novel chromatin regulators. After characterizing thousands of domains drawn from all the different chromatin regulatory complexes, we will create and share a detailed resource of compact and efficient domains that can be fused onto CRISPR DNA- binding proteins in order to recruit desired chromatin regulatory complexes to act upon a genomic element. In order to positively impact human health with these approaches, I propose to leverage this training to develop new methods that dissect transcriptional dysregulation in kidney disease during the postdoctoral phase.

染色质修饰涉及人类细胞中所有基本的DNA模板化过程，包括 转录，并在许多疾病中被错误调节。通过CRISPR靶向技术，我们可以写 特定的染色质修饰，然后测量基因表达如何响应。这将使 我们了解基因表达如何在疾病状态下变化，以及如何合理设计治疗方法， 扭转这些变化。 使用CRISPR扰动来研究非编码调控元件的挑战之一是CRISPR关闭。 目标活动。在这里，我们表明非编码扰动实验中的脱靶效应可以与 在人类细胞中具有显著的毒性，不仅具有DNA切割Cas9，而且具有表观基因组修饰 CRISPRi/a工具。在去除了容易脱靶的引导RNA之后，我们可以使用CRISPR来精确地连接非编码RNA。 基因调控元件。 这些扰动实验对于了解染色质修饰的因果函数至关重要， 特定的基因组元素。然而，迄今为止，大多数实验都使用CRISPRi，其中一个特定的KRAB 结构域建立一个特定的异染色质状态。操纵染色质状态的现有工具是 大部分来自数千种天然染色质调节复合物中的一小部分;大多数患有 部分或短暂的影响，并表现出跨基因座和细胞类型的高变异性。更完整的工具箱， 紧凑，有效的结构域的途径特异性染色质扰动将改变我们的能力，以确定 人类基因组中特定染色质修饰的因果作用。在此，我们建议 系统地测量将染色质调节蛋白结构域募集到细胞中的基因表达效应， 启动子这是可能的，我们最近开发的高通量染色质调节剂招募 在人细胞中的测定，能够同时测量数万个调节结构域的活性。 使用这个系统，我们将从启动子中招募和释放CR变体，然后测量其大小。 和在报告基因座处转录沉默的持久性。然后，我们将使用表观基因组作图分析， 确定染色质修饰，这些新的染色质调节沉默功能的基础。 在表征了来自所有不同染色质调节复合物的数千个结构域之后，我们将 创建和共享可以融合到CRISPR DNA上的紧凑高效结构域的详细资源- 结合蛋白，以募集所需的染色质调节复合物作用于基因组元件。 为了用这些方法对人类健康产生积极影响，我建议利用这一培训， 开发新的方法，在博士后阶段解剖肾脏疾病的转录失调。