Development of functional genomic technologies in mice

小鼠功能基因组技术的发展

基本信息

批准号：
10506700
负责人：
Aki Ushiki
金额：
$ 11.39万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-01-01 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10506700
关键词：
ATAC-seq Address Biological Assay Brain CRISPR screen Catalogs Cells ChIP-seq Chromosome Mapping Clustered Regularly Interspaced Short Palindromic Repeats Code Color Complex Cultured Cells DNA DNA Sequence DNA Transposable Elements Data Dependovirus Development Disease Ectoderm Electroporation Elements Embryo Endoderm Enhancers Ensure Female Future Genetic Enhancer Element Genome Genomics Germ Layers Glycoproteins Goals Guide RNA Heart Heterozygote Human Genome In Vitro Individual Knock-out Lead Libraries Liver Mammalian Oviducts Measures Mentors Mesoderm Methods Mus Mutagenesis Mutation Nucleic Acid Regulatory Sequences Nucleotides Organism Penetration Phase Phenotype Publications Regulator Genes Regulatory Element Reporter Reporting Research Research Personnel Serotyping Single Nucleotide Polymorphism Specificity System Techniques Technology Testing Thick Time Tissues Training Transgenic Mice Transposase Untranslated RNA Variant Zona Pellucida base editor career cell type ds-DNA extracellular functional genomics gain of function gastrulation gene function genome wide association study genome-wide genomic data genomic tools high throughput screening high throughput technology human disease in vivo loss of function novel pregnant prevent prime editor promoter screening tool zygote

项目摘要

PROJECT SUMMARY Mutations in gene regulatory elements (REs) are a major cause of human disease. For example, the majority (>90%) of disease related genome wide association studies (GWAS) found associations with variants in non- coding and likely regulatory regions in the genome. Despite their importance, the code and grammar of these regulatory elements remains largely unknown making the understanding of how mutations in these sequences can lead to disease even more complex. Regulatory elements can be identified in a genome-wide manner using techniques such as ChIP-seq or ATAC-seq. However, these methods are descriptive and do not provide a functional readout that tests whether these elements are indeed functional. Massively parallel reporter assays (MPRAs) and CRISPR-based screens have recently been developed to functionally characterized these elements in a high-throughput manner. However, most of these techniques use cultured cells to measure activity. As such, the activity and function of these elements and their variants in an organism has not been tested. Due to this, complex phenotypes, such as spatial-temporal, and tissue/cell type specificity and interactions cannot be assessed for these elements. In this K99/R00 application, I will develop technologies that will allow to functionally characterize regulatory elements and variants in a high throughput manner in mice. One of the biggest barriers that prevent high-throughput assays in mice is the zona pellucida that surrounds one-cell stage embryos and prevents double-stranded DNA to be inserted. Recent reports and my own preliminary data show that by utilizing adeno associated virus serotype 6 (AAV6) as a delivery tool, DNA can integrate into one-cell stage embryos. I plan to use AAV6 along with the PiggyBac transposase system, that allows for genomic integration in all three-germ layers, to develop MPRA in mice (Aim K1). To validate the effect of single nucleotide variants, I will develop large-scale CRISPR saturation mutagenesis assays in mice. This will be done by utilizing in vitro electroporation into embryos and base-editor or prime-editor transgenic mice (Aim K2). Finally, I will apply these technologies to generate a catalog of functional regulatory elements, including transposable elements, involved in differentiation of the three primary germ layers (Aim R1). The results from this proposal will provide novel in vivo high-throughput technologies that will enable to study regulatory elements and disease-associated variants at any developmental time stage in mice. My career goal is to lead an independent research group developing novel functional genomics tools in mice and studying the function of gene regulatory elements and their variants in tissue development and disease utilizing these technologies. To achieve this goal, I will receive experimental and computational training from my mentors Drs. Nadav Ahituv and Jay Shendure. This rigorous mentored support and results obtained in the K99 phase will ensure my transition to an independent investigator and future successful independent career.

项目概要基因调控元件（RE）突变是人类疾病的主要原因。例如，大多数 (>90%) 的疾病相关全基因组关联研究 (GWAS) 发现与非基因变异的关联基因组中的编码区和可能的调控区。尽管它们很重要，但它们的代码和语法调控元件在很大程度上仍然未知，因此需要了解这些序列中的突变是如何发生的可能导致疾病更加复杂。可以以全基因组的方式识别调控元件使用 ChIP-seq 或 ATAC-seq 等技术。然而，这些方法都是描述性的，并没有提供功能读数，用于测试这些元件是否确实具有功能。大规模并行报告器最近已开发出基于 CRISPR 的分析（MPRA）和筛选来进行功能表征以高通量的方式处理这些元素。然而，大多数这些技术使用培养细胞来测量活动。因此，这些元素及其变体在生物体中的活性和功能尚未得到证实。已测试。因此，复杂的表型，例如时空、组织/细胞类型特异性和无法评估这些元素的相互作用。在这个K99/R00应用中，我将开发技术这将允许以高通量方式对调控元件和变体进行功能表征老鼠。阻碍小鼠高通量检测的最大障碍之一是透明带包围单细胞阶段胚胎并阻止双链 DNA 插入。最近的报告和我的我们自己的初步数据表明，通过利用腺相关病毒血清型 6 (AAV6) 作为传递工具，DNA 可以整合到单细胞阶段胚胎中。我计划将 AAV6 与 PiggyBac 转座酶系统一起使用，允许在所有三胚层中进行基因组整合，从而在小鼠中开发 MPRA（Aim K1）。为了验证为了研究单核苷酸变异的影响，我将在小鼠中开发大规模 CRISPR 饱和诱变试验。这将通过利用体外电穿孔进入胚胎和碱基编辑器或初等编辑器转基因来完成小鼠（目标 K2）。最后，我将应用这些技术来生成功能调控元件的目录，包括参与三个初级胚层分化的转座因子（Aim R1）。这该提案的结果将提供新颖的体内高通量技术，使研究小鼠任何发育时间阶段的调控元件和疾病相关变异。我的职业目标领导一个独立研究小组在小鼠中开发新型功能基因组学工具并研究利用这些基因调控元件及其变体在组织发育和疾病中的功能技术。为了实现这一目标，我将接受我的导师 Drs. 的实验和计算培训。纳达夫·阿希图夫和杰伊·申杜尔。这种严格的指导支持和在 K99 阶段获得的结果将确保我向独立调查员的过渡以及未来成功的独立职业生涯。