Glycome-Enhanced KnockOut (GEKO) Technology

 说明(申请人提供)：糖组学领域是一个具有技术挑战性的领域，通常使用专门的试剂和设备，因此限制了其在二次影响领域的广泛采用。此外，许多进展强调了化学合成、大分子相互作用和改进的质谱学方法，这推动了我们对该领域的生化理解，但对降低外部研究人员的进入门槛几乎没有起到什么作用。到期 由于没有扩展到其他领域，糖糖对体内生物学的影响以及我们对糖糖如何适应疾病机制的了解仍处于萌芽阶段。为了弥补这一差距，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来探索糖苷在任何生物系统或途径中的影响，我们认为前进的道路是提供适用于小鼠和人类生物学的简便基因技术，而不需要专门的专业知识。因此，我们建议利用基于CRISPR的分子生物学来创建糖糖激活敲除(GEKO)靶向技术，以选择性地消融跨多个物种的糖糖相关基因。建议的GEKO技术的主要成果是：(1)100个针对50个遗传位点的有效CRISPR靶向构建体，用于选择性切割糖类相关基因；(2)用于灵活地将CRISPR技术应用于其他系统的有效靶向序列；以及(3)50个人类HEK293 GEKO细胞系，每个糖类相关基因经过验证的敲除。这些工具将使糖生物学家和非糖生物学家能够在体外、体外和体内通过遗传学轻松地操纵糖糖；从而提供一个廉价的途径，在从癌症和神经退化到细胞内信号和免疫的各种正常和疾病背景下探索多糖对蛋白质、细胞和组织功能的影响。

 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.