Glycome-Enhanced KnockOut (GEKO) Technology

糖类增强敲除 (GEKO) 技术

• 批准号: 9108958
• 负责人: Derek W Abbott
• 金额: $ 30.75万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-10 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9108958
• 关键词: Ablation; Address; Adoption; American; Awareness; Biochemical; Biology; CRISPR/Cas technology; 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; Health; Histocompatibility Testing; Human; Human Biology; Immunity; Immunology; In Vitro; Knock-in Mouse; Knock-out; Knockout Mice; 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; sialylation; tool

 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，从而限制了其在二级影响领域的广泛采用。此外，许多进展都强调化学合成，大分子相互作用和改进的质谱方法，这推动了我们对生物化学的理解，但也没有降低外部研究人员的进入门槛。由于 由于缺乏扩展到其他领域，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认识仍处于其初期阶段。为了解决这一差距，我们必须努力为生物医学研究人员提供一套更完整的工具，以探索糖组在任何生物系统或途径中的影响，我们认为，前进的方向是提供适用于小鼠和人类生物学的简易遗传技术，而无需专业知识。因此，我们提出利用基于CRISPR的分子生物学来创建糖组启用敲除（GEKO）靶向技术，用于跨多个物种选择性地消融糖组相关基因。所提出的GEKO技术的主要可交付成果是：（1）100个经验证的CRISPR靶向构建体，靶向50个遗传基因座，用于选择性消融糖组相关基因，（2）经验证的靶向序列，用于将CRISPR技术敏捷应用于其他系统，以及（3）50个人HEK 293 GEKO细胞系，每个糖组相关基因均经验证敲除。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地操纵糖组;从而提供一个廉价的途径来探索聚糖对蛋白质、细胞和组织功能的影响，这些影响涉及从癌症和神经变性到细胞内信号传导和免疫的各种正常和疾病背景。