Genome-wide Analysis of Heparan Sulfate using CRISPR/Cas9

 描述（由申请人提供）：动物细胞含有多种硫酸乙酰肝素蛋白聚糖（HSPG），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结合许多生长因子、细胞因子、细胞外基质组分、酶和酶抑制剂，并且它们充当细胞信号传导、细胞附着和内吞作用的受体或共受体。这些相互作用在很大程度上取决于硫酸乙酰肝素链中硫酸化糖和糖醛酸差向异构体的组成和排列，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、进行生物化学研究以及在细胞或模式生物中突变。与此相反，缺乏信息存在的调节因素，引起的可变组成和结合特性的硫酸乙酰肝素。该提议的中心假设是，存在编码生物合成酶的基因以外的基因，其表达调节酶的转录/翻译或协调其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靶向技术来寻找这些调控因子。为了实现这一目标，我们将（i）采用高通量筛选测定来鉴定慢病毒编码的sgRNA，其诱导对细胞毒性剂的抗性，所述细胞毒性剂的作用依赖于HSPG（FGF-皂草素和白喉毒素）;（ii）在系统水平上鉴定、优先化和分析从筛选中出现的基因，和（iii）在三种不同细胞系中靶向优先化的基因以了解它们在调节HSPG表达中的作用模式。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发现新的参与HSPG表达的调节因子的潜力。这些实验的结果有一个更广泛的目标，那就是将该领域转移到新的研究领域。深入了解细胞用于调节硫酸乙酰肝素成分的机制也可能导致新的药物靶点，用于治疗与硫酸乙酰肝素形成改变相关的人类疾病，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.