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 基因调节元件（RES）的突变是人类疾病的主要原因。例如，大多数 （> 90％）与疾病相关的基因组广泛关联研究（GWAS）发现与非 - 基因组中的编码和可能的调节区域。尽管它们的重要性，但这些代码和语法 监管元素在很大程度上是未知的，使得了解这些序列中的突变如何 可能导致疾病更加复杂。可以以全基因组的方式识别监管元素 使用诸如chip-seq或atac-seq之类的技术。但是，这些方法具有描述性，并且不提供 一个功能读数，可以测试这些元素是否确实功能正常。大量平行的记者 最近已经开发了用于功能表征的测定（MPRA）和基于CRISPR的屏幕 这些元素以高通量方式。但是，这些技术中的大多数都使用培养的细胞来测量 活动。因此，这些元素的活动和功能及其在生物体中的变体尚未 测试。因此，复杂的表型，例如时空，组织/细胞类型特异性以及 这些要素无法评估相互作用。在此K99/R00应用程序中，我将开发技术 这将允许以高吞吐量的方式在功能上表征调节元素和变体 老鼠。防止小鼠高通量测定的最大障碍之一是Zona Pellucida 包围一个单细胞阶段的胚胎，并防止插入双链的DNA。最近的报告和我的 自己的初步数据表明，通过利用Adeno相关病毒血清型6（AAV6）作为递送工具，DNA 可以集成到一个单细胞阶段的胚胎中。我计划将AAV6与PiggyBac转座酶系统一起使用， 这允许在所有三粒层中进行基因组整合，以在小鼠中发展MPRA（AIM K1）。验证 单核苷酸变体的影响，我将在小鼠中开发大规模的CRISPR饱和诱变测定。 这将通过利用体外电穿孔到胚胎和基本编辑器或原始编辑转基因来完成 小鼠（AIM K2）。最后，我将应用这些技术生成功能调节元素的目录， 包括转座元素，涉及三个主要细菌层的分化（AIM R1）。这 该提案的结果将提供新颖的体内高通量技术，这些技术将能够研究 小鼠的任何发育时间阶段的调节元件和疾病相关的变体。我的职业目标 是领导一个独立的研究小组，开发小鼠中新型功能基因组学工具并研究 基因调节元素及其在组织发育和疾病中的变异的功能利用这些功能 技术。为了实现这一目标，我将从导师DRS获得实验和计算培训。 纳达夫·阿希图夫和杰伊·史德尔。这种严格的指导支持和在K99阶段获得的结果将 确保我过渡到独立研究者和未来成功的独立职业。