Genome-wide Analysis of Heparan Sulfate using CRISPR/Cas9

使用 CRISPR/Cas9 对硫酸乙酰肝素进行全基因组分析

• 批准号: 9103016
• 负责人: Jeffrey D Esko
• 金额: $ 16.86万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9103016
• 关键词: Accounting; Affect; Animal Model; Animals; Area; Atherosclerosis; Binding; Biogenesis; Biological Assay; CRISPR screen; CRISPR/Cas technology; Candidate Disease Gene; Categories; Cell Adhesion; Cell Line; Cell-Matrix Junction; Cells; Collection; Core Protein; Cytotoxic agent; Cytotoxin; DNA; Data; Development; Diphtheria Toxin; Disease; Endocytosis; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Extracellular Matrix; Fibroblast Growth Factor; Future; Gene Expression; Gene Silencing; Gene Targeting; Genes; Genetic; Genetic Transcription; Goals; Growth; Guide RNA; Harvest; Health; Heparan Sulfate Biosynthesis; Heparan Sulfate Proteoglycan; Heparitin Sulfate; Human Genome; Individual; Induced Mutation; Inflammation; Inorganic Sulfates; Lead; Lentivirus Vector; Ligand Binding; Ligands; Link; Malignant Neoplasms; Methods; Modification; Mutate; Organ; Outcome; Pathway interactions; Pattern; Physiology; Plant Lectins; Plasmids; Polysaccharides; Property; Proteins; RNA Sequences; RNA library; Recombinant Proteins; Resistance; Sorting - Cell Movement; Substrate Specificity; System; Systems Biology; Techniques; Testing; Tissues; Toxin; Translations; Unspecified or Sulfate Ion Sulfates; Uronic Acids; Work; base; cell type; cytokine; deep sequencing; drug development; genome editing; genome-wide; genome-wide analysis; glycosylation; high throughput screening; human disease; insight; intercellular communication; new therapeutic target; novel; overexpression; receptor; research study; screening; sugar; transcriptome sequencing; whole genome

 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.

 描述（申请人提供）：动物细胞精制出多种硫酸乙酰肝素蛋白聚糖（HSPG），其由不同的蛋白核心和一条或多条硫酸乙酰肝素链组成。 HSPG 结合多种生长因子、细胞因子、细胞外基质成分、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、进行生化研究，并在细胞或模型生物体中发生突变。相比之下，关于导致硫酸乙酰肝素的不同组成和结合特性的调节因素的信息却很缺乏。该提议的中心假设是，除了那些编码生物合成酶的基因之外，还存在其他基因，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。我们的目标是使用 CRISPR/Cas9 基因组编辑系统通过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鉴定慢病毒编码的 sgRNA，这些 sgRNA 可诱导细胞毒性药物的抗性，而细胞毒性药物的作用依赖于 HSPG（FGF-肥皂草素和白喉毒素）； (ii) 在系统水平上识别、优先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的优先基因，以了解它们在调节 HSPG 表达方面的作用模式。还进行了 HS 的详细结构研究以及与测试配体的结合研究。这项工作的意义在于发现参与 HSPG 表达的新调控因子的潜力。这些实验的结果具有更广泛的目标，即将该领域转移到新的研究领域。对细胞调节硫酸乙酰肝素组成的机制的深入了解也可能会产生新的药物靶点，用于治疗与硫酸乙酰肝素形成改变相关的人类疾病，例如癌症、炎症和动脉粥样硬化。

项目成果

Author Correction: Genome-wide screens uncover KDM2B as a modifier of protein binding to heparan sulfate.

作者更正：全基因组筛选发现 KDM2B 作为蛋白质与硫酸乙酰肝素结合的修饰剂。

• DOI: 10.1038/s41589-022-01022-6
• 发表时间: 2022
• 期刊: Nature chemical biology
• 影响因子: 14.8
• 作者: Weiss,RyanJ;Spahn,PhilippN;Chiang,AustinWT;Liu,Qing;Li,Jing;Hamill,KristinaM;Rother,Sandra;Clausen,ThomasM;Hoeksema,MartenA;Timm,BryceM;Godula,Kamil;Glass,ChristopherK;Tor,Yitzhak;Gordts,PhilipLSM;Lewis,NathanE;E
• 通讯作者: E