High-throughput dissection of transcriptional regulation in kidney disease

染色质修饰参与了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控。使用CRISPR目标定位技术，我们可以编写 具体的染色质修饰，然后测量基因表达的变化。这将使 美国需要了解疾病状态下基因表达的变化，以及如何合理设计治疗方法 逆转这些变化。 使用CRISPR扰动研究非编码调控元件的挑战之一是CRISPR OFF- 目标活动。在这里，我们证明了非编码扰动实验中的脱靶效应可以关联 对人类细胞有显著的毒性，不仅有DNA裂解的Cas9，而且还有表观基因组的修饰 CRISPRi/a工具。在去除容易偏离目标的引导RNA后，我们可以使用CRISPR来准确链接非编码 与基因有关的调控元件。 这些微扰实验对于了解染色质修饰的因果函数至关重要 特定的基因组成分。然而，到目前为止，大多数实验都使用了CRISPRi，有一个特殊的KRAB 建立一种特殊的异染色质状态的结构域。现有的处理染色质状态的工具有 主要来自数千种天然染色质调节复合体中的一小部分；大多数患有 部分或瞬时效应，并且在不同的基因座和细胞类型上表现出高度的可变性。更完整的工具箱 途径特异性染色质扰动的紧凑、有效的结构域将改变我们确定 人类基因组中特定染色质修饰的因果作用。在此，我们建议 系统检测重组染色质调节蛋白结构域对基因表达的影响 推动者。这是由于我们最近开发的高通量染色质调节器招募 在人体细胞中进行检测，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 以及在报道地点转录沉默的永久性。然后我们将使用表观基因组图谱分析来 确定支持这些新型染色质调节剂沉默功能的染色质修饰。 在鉴定了来自所有不同染色质调节复合体的数千个结构域之后，我们将 创建和共享可融合到CRISPR DNA上的紧凑高效域的详细资源- 结合蛋白质，以招募所需的染色质调节复合体作用于基因组元件。 为了通过这些方法积极影响人类健康，我建议利用这次培训来 开发新的方法，在博士后阶段剖析肾脏疾病的转录失调。

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.