Development of functional genomic technologies in mice

小鼠功能基因组技术的发展

• 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
• 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

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.

项目摘要 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%）与疾病相关的全基因组关联研究（GWAS）发现与非基因组变异相关。 基因组中的编码区和可能的调控区。尽管它们的重要性，这些代码和语法 调控元件在很大程度上仍然是未知的，使得理解这些序列中的突变如何 会导致更复杂的疾病调控元件可以在全基因组范围内进行鉴定 使用诸如ChIP-seq或ATAC-seq的技术。然而，这些方法是描述性的，不提供 测试这些元素是否真的起作用的功能读出器。大规模并行报告程序 最近已经开发了基于MPRAs和CRISPR的筛选来功能性地表征 这些元素以高通量的方式。然而，大多数这些技术使用培养的细胞来测量 活动因此，这些元件及其变体在生物体中的活性和功能尚未被阐明。 测试.因此，复杂的表型，如时空和组织/细胞类型特异性， 不能评估这些元素的相互作用。在此K99/R 00应用程序中，我将开发技术 这将允许以高通量的方式在功能上表征调节元件和变体， 小鼠阻止小鼠中高通量测定的最大障碍之一是透明质酸， 包围着单细胞阶段的胚胎并阻止双链DNA的插入。最近的报告和我 自己的初步数据显示，通过利用腺相关病毒血清型6（AAV 6）作为递送工具，DNA 可以整合到单细胞阶段的胚胎中。我计划使用AAV 6沿着PiggyBac转座酶系统， 允许基因组整合在所有三个胚层中，以在小鼠中开发MPRA（Aim K1）。验证 为了研究单核苷酸变体的影响，我将在小鼠中开发大规模的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或引物编辑转基因来完成。 小鼠（Aim K2）。最后，我将应用这些技术来生成功能性调节元件的目录， 包括转座因子，参与三个初级胚层的分化（Aim R1）。的 该建议的结果将提供新的体内高通量技术， 调节元件和疾病相关的变体在小鼠的任何发育时间阶段。我的职业目标 是领导一个独立的研究小组，在小鼠中开发新的功能基因组学工具，并研究 基因调控元件及其变体在组织发育和疾病中的功能 技术.为了实现这个目标，我将从我的导师Dr。 Nadav Ahituv和Jay Shendure。这种严格的指导支持和在K99阶段获得的结果将 确保我过渡到独立调查员和未来成功的独立职业生涯。