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.

项目总结 基因调控元件(RES)的突变是人类疾病的主要原因。例如，大多数人 (&gt；90%)的疾病相关全基因组关联研究(GWAS)发现与非 基因组中的编码和可能的调节区。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是未知的，这使得我们了解这些序列中的突变是如何 会导致更复杂的疾病。可以在全基因组范围内识别调控元件 使用诸如CHIP-SEQ或ATAC-SEQ等技术。但是，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数。大规模平行记者 分析(MPRA)和基于CRISPR的屏幕最近已被开发用于功能表征 这些元素以高吞吐量的方式。然而，这些技术中的大多数都是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动和功能尚未被 测试过。因此，复杂的表型，如时空和组织/细胞类型特异性和 无法评估这些元素的交互作用。在这个K99/R00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控元件和变体 老鼠。阻碍小鼠高通量检测的最大障碍之一是透明带，它 包围单细胞期胚胎，防止双链DNA插入。最近的报告和我的 自身的初步数据表明，通过使用腺相关病毒6型(AAV6)作为递送工具，DNA 可以整合成单细胞期的胚胎。我计划将AAV6与PiggyBac转座酶系统一起使用， 这允许在所有三种胚层中进行基因组整合，以在小鼠身上开发MPRA(目标K1)。要验证 在单核苷酸变异的影响下，我将在小鼠中开展大规模CRISPR饱和突变试验。 这将通过利用体外电穿孔进入胚胎和碱基编辑或初级编辑转基因来完成 小鼠(靶标K2)。最后，我将应用这些技术来生成功能监管要素的目录， 包括转座子，参与三个初级胚层的分化(目标R1)。这个 这项提议的结果将提供新的体内高通量技术，使研究 在小鼠的任何发育阶段的调控元件和疾病相关变异。我的职业目标 领导一个独立的研究小组在小鼠身上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作用 技术。为了实现这一目标，我将接受我的导师Dr。 纳达夫·阿希图夫和杰伊·申杜尔。在K99阶段中获得的这种严格的指导支持和结果将 确保我转型为一名独立调查员，并在未来取得成功的独立职业生涯。