Genome-wide Analysis of Heparan Sulfate using CRISPR/Cas9

 描述(申请人提供)：动物细胞精制各种硫酸乙酰肝素蛋白多糖(HSPGs)，由不同的蛋白质核心和一条或多条硫酸乙酰肝素链组成。HSPG结合了大量的生长因子、细胞因子、细胞外基质成分、酶和酶抑制剂，它们作为细胞信号、细胞附着和内吞的受体或辅助受体。这些相互作用在很大程度上取决于硫酸乙酰肝素链上糖醛酸的组成和排列，而这又取决于各种生物合成酶和调节因子的作用。大多数参与硫酸乙酰肝素生物合成的酶已被鉴定、克隆、表达为重组蛋白、生化研究，并在细胞或模式生物中发生突变。相反，缺乏关于引起硫酸乙酰肝素不同组成和结合特性的调节因素的信息。这一建议的中心假设是，除了那些编码生物合成酶的基因外，还有其他基因存在，这些基因的表达要么调节酶的转录/翻译，要么协调它们的作用，以实现在不同细胞类型中观察到的硫酸乙酰肝素的最终组成。我们的目标是通过使用CRISPR/Cas9基因组编辑系统的全基因组基因打靶技术来寻找这些调控因子。为了实现这一目标，我们将(I)采用高通量筛选方法来识别慢病毒编码的sgRNA，这些sgRNA诱导对其作用依赖于HSPGs(成纤维细胞生长因子-Saporin和白喉毒素)的细胞毒剂产生抗药性；(Ii)在系统水平上识别、排序和分析从筛选中出现的基因，以及(Iii)针对三种不同细胞系中优先考虑的基因，以了解它们在调节HSPG表达方面的作用模式。还进行了HS的详细结构研究和与测试配体的结合研究。这项工作的意义在于有可能发现与HSPG表达有关的新的调节因子。这些实验的结果有一个更广泛的目标，那就是将该领域带入新的研究领域。深入了解细胞用来调节硫酸肝素成分的机制也可能导致治疗与硫酸肝素形成变化相关的人类疾病的新药物靶点，如癌症、炎症和动脉粥样硬化。

 DESCRIPTION (provided by applicant): Animal cells elaborate a variety of heparan sulfate proteoglycans (HSPGs), which consist of different protein cores and one or more heparan sulfate chains. The HSPGs bind numerous growth factors, cytokines, extracellular matrix components, enzymes and enzyme inhibitors, and they act as receptors or coreceptors for cell signaling, cell attachment, and endocytosis. These interactions depend to a large extent on the composition and the arrangement of sulfated sugars and epimers of uronic acids in the heparan sulfate chains, which in turn depend on the action of various biosynthetic enzymes and regulatory factors. Most of the enzymes involved in heparan sulfate biogenesis have been identified, cloned, expressed as recombinant proteins, studied biochemically, and mutated in cells or model organisms. In contrast, a dearth of information exists regarding the regulatory factors that give rise to the variable composition and binding properties of heparan sulfate. The central hypothesis of this proposal is that genes exist other than those that encode the biosynthetic enzymes, whose expression either modulate the transcription/translation of the enzymes or orchestrate their action to achieve the final composition of heparan sulfate observed in different cell types. Our objective is to search for these regulatory factors through whole genome gene-targeting techniques using the CRISPR/Cas9 genome editing system. Towards this goal, we will (i) adapt high throughput screening assays to identify lentiviral encoded sgRNAs that induce resistance to cytotoxic agents whose action depend on HSPGs (FGF-Saporin and diphtheria toxin); (ii) identify, prioritize, and analyze at the systems level genes tht emerge from the screen, and (iii) target prioritized genes in three different cell lines to understand their mode of action in regulating HSPG expression. Detailed structural studies of HS and binding studies with test ligands also are performed. The significance of this work lies in the potential for discovery of novel regulatory factors involved in HSPG expression. The outcome of these experiments has the wider goal of moving the field into new areas of study. Insight into the mechanisms that cells use to regulate heparan sulfate composition also might lead to novel drug targets for treating human disease associated with alterations in heparan sulfate formation, such as cancer, inflammation and atherosclerosis.