Glycome-Enhanced KnockOut (GEKO) Technology

糖类增强敲除 (GEKO) 技术

基本信息

批准号：
8985066
负责人：
Derek W Abbott
金额：
$ 30.01万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-10 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8985066
关键词：
Ablation Address Adoption American Awareness Biochemical Biology Cell Line Cells Chinese Hamster Chinese Hamster Ovary Cell Clustered Regularly Interspaced Short Palindromic Repeats Collection Communities Complex Defect Derivation procedure Development Disease Dissection Enzymes Equipment Flow Cytometry Funding Genes Genetic Grant Growth Histocompatibility Testing Human Human Biology Immunity Immunology In Vitro Knock-out Knowledge Lectin Libraries Malignant Neoplasms Mass Spectrum Analysis Methodology Molecular Biology Mus Mutation Nerve Degeneration Ovary Pathway interactions Phase Polysaccharides Population Protein-Carbohydrate Interaction Proteomics Reagent Recombinant Proteins Renaissance Research Research Personnel Role Series Signal Transduction Sorting - Cell Movement Source Subfamily lentivirinae System Techniques Technology Time Tissues Western Blotting Work anticancer research arm base biological systems chemical synthesis cost field study genetic manipulation genetic technology glycosylation immortalized cell improved in vivo instrumentation interest new technology novel protein function public health relevance sialylation targeted sequencing tool

项目摘要

DESCRIPTION (provided by applicant): The field of glycomics is a technically challenging field which commonly utilizes specialized reagents and equipment, thereby limiting its widespread adoption in secondary fields of impact. Moreover, many of the advances have emphasized chemical synthesis, macromolecular interactions, and improved mass spectroscopy methodology, which has pushed the field forward in terms of our biochemical understanding but has also done little to lower the barrier of entry for outside investigators. Due to the lack of expansion into other fields, the impact of the glycome on in vivo biology and our knowledge of how the glycome fits into disease mechanisms remains in its nascent phase. To address this gap, we must strive to provide a more complete set of tools for biomedical investigators to explore the influence of the glycome in any biological system or pathway, and we believe that the way forward is to provide facile genetic technology applicable to both murine and human biology without the requirements for specialized expertise. We therefore propose to utilize CRISPR-based molecular biology to create Glycome-Enabled KnockOut (GEKO) targeting technology for the selective ablation of glycome- associated genes across multiple species. The primary deliverables from the proposed GEKO technology are: (1) 100 validated CRISPR targeting constructs targeting 50 genetic loci for the selective ablation of glycome- associated genes, (2) validated targeting sequences for agile application of the CRISPR technology to other systems, and (3) 50 human HEK293 GEKO cell lines with validated knockout of each glycome-associated gene. These tools will enable glycobiologists and non-glycobiologists alike to easily manipulate the glycome through genetics in vitro, ex vivo, and in vivo; thereby providing an inexpensive gateway to explore the influence of glycans on the function of proteins, cells, and tissues in a wide variety of normal and disease contexts ranging from cancer and neurodegeneration to intracellular signaling and immunity.

描述（由申请人提供）：糖组学领域是一个技术上具有挑战性的领域，通常使用专门的试剂和设备，从而限制了其在次要影响领域的广泛采用。此外，许多进展都强调化学合成、大分子相互作用和化学合成。改进的质谱方法，在我们的生化理解方面推动了该领域的发展，但也几乎没有降低外部研究人员的进入门槛。由于缺乏向其他领域的扩展，糖组对体内生物学的影响以及我们对糖组如何适应疾病机制的了解仍处于初级阶段。为了弥补这一差距，我们必须努力提供一套更完整的研究。生物医学研究人员探索糖组在任何生物系统或途径中的影响的工具，我们相信前进的方向是提供适用于小鼠和人类生物学的简便遗传技术，而无需专业知识，因此我们建议利用。基于 CRISPR 的分子生物学创建 Glycome-Enabled KnockOut (GEKO) 靶向技术，用于选择性消除多个物种的糖组相关基因。拟议的 GEKO 技术的主要成果是：(1) 100 个经过验证的 CRISPR 靶向构建体，针对 50 个基因位点，用于选择性消除糖组相关基因。糖组相关基因，(2) 经过验证的靶向序列，以便将 CRISPR 技术灵活应用于其他系统，以及 (3) 50 个人类 HEK293 GEKO这些工具将使糖生物学家和非糖生物学家能够通过体外、离体和体内遗传学轻松操纵糖组，从而为探索聚糖的影响提供一种廉价的途径；研究蛋白质、细胞和组织在各种正常和疾病环境中的功能，从癌症和神经变性到细胞内信号传导和免疫。