High content glycomics analysis using next generation sequencing technology

使用下一代测序技术进行高内涵糖组学分析

• 批准号: 9924616
• 负责人: SRIRAM NEELAMEGHAM
• 金额: $ 23.81万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9924616
• 关键词: Address; Anabolism; Antigens; Area; Bar Codes; Basic Science; Binding; Biochemical; Biochemical Pathway; Biochemical Reaction; Bioinformatics; Biological; Biological Assay; Biology; Blood; Blood Cells; Breast; Breast Cancer Cell; Breast Cancer cell line; CRISPR library; CRISPR screen; CRISPR/Cas technology; Cancer Biology; Carbohydrates; Cardiovascular Diseases; Catalogs; Cell Line; Cell physiology; Cells; Chemistry; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Communities; Complex Mixtures; Computer Simulation; Cytometry; Data; Data Analyses; Development; Diagnosis; Disease model; Epitopes; Fixatives; Flow Cytometry; Future; Genes; Genetic Screening; Goals; Human; Individual; Investigation; Knock-out; Label; Lead; Lectin; Libraries; Literature; Malignant Neoplasms; Mammalian Cell; Mammary Gland Parenchyma; Mammary Neoplasms; Maps; Measurement; Measures; Methodology; Methods; MicroRNAs; Molecular; Monitor; Monoclonal Antibodies; Neurosciences; Oligonucleotides; Output; Pathologic; Pathology; Pathway Analysis; Pathway interactions; Patient Care; Patients; Phenotype; Physiology; Polysaccharides; Population; Preparation; Procedures; Process; Property; Reagent; Regenerative Medicine; Research; Sampling; Selection Criteria; Series; Specificity; Specimen; System; Technology; Testing; Tissues; Transcriptional Regulation; Transition Elements; Viral; base; biological systems; carbohydrate binding protein; carbohydrate biosynthesis; carbohydrate structure; cell type; computerized tools; density; design; experimental study; fluorophore; functional loss; genome editing; genome-wide; glycosylation; human tissue; interest; knockout gene; mathematical analysis; mathematical model; next generation sequencing; novel; preservation; response; scale up; tool; web site; whole genome

Biological systems are typically robust since many different biochemical reaction pathways and processes partially control system stability. In some instances, loss of a single pathway results in compensatory contributions from others to maintain system-status. In others, many different biological pathways partially contribute to regulating a single function, with complete functional loss only being observed upon loss or knocking-out of all individual contributors. Large-scale experimental and computational interrogation of biological assemblies is necessary in order to develop a systems-level understanding. The scope of such investigations has been vastly enhanced in recent years with the development of new multiplex tools based on next-generation sequencing (NGS). This is commonly enabled by the tagging of individual cells, monoclonal antibodies or other antigen binders with oligonucleotide barcodes or unique molecular identifiers (UMIs). Such barcodes/UMIs can be followed independently using NGS. In addition, it is now possible to develop CRISPR based knockout libraries and screens targeting the entire genomes, or a subset of genes related to a certain function (e.g. cellular glycosylation). This project aims to combine recent advances in NGS and CRISPR-Cas9 by addressing the hypothesis that ‘the combined use of CRISPR-Cas to knockout genes in large scale combined with NGS to measure related cell-system response can inform us of system properties at an unprecedented scale’. We test this possibility using examples from the Glycosciences. The specific aims are: Aim 1. To develop ‘Lectin-Tag- seq’, a method to assay the glycome of individual cells in complex mixtures. Here, a panel of lectins and related mAbs are tagged with uniquely barcoded oligonucleotides. The simultaneous binding of these reagents to diverse cell types in human blood and breast tissue is measured at a large scale using NGS. The lectin binding specificity of a vast number of carbohydrate binding proteins is now available in literature, and thus bioinformatics analysis will be applied to relate the lectin-binding measurements on individual cells to glycan epitopes and potential carbohydrate structures that exist on the single cells. Aim 2. To develop ‘CRISPR-Tag-seq’ in order to simultaneously measure, at a single cell level, the sgRNA editing a given cell and the corresponding changes in multiplex lectin/mAb binding profiles. Here, a glycogene-CRISPR library targeting 347 genes regulating cellular glycosylation is introduced into breast cancer cells. The effects of such biochemical pathway perturbations are related to lectin binding on individual cells. Mathematical analysis is performed to analyze the effect of sgRNA perturbations. All data will be available to the research community at our website VirtualGlycome.org. In the long run, methods developed in this project can be extended to additional cell types and biological problems to ultimately streamline and simplify our understanding of the mammalian glycome.

生物系统通常是稳健的，因为许多不同的生化反应途径和过程 部分控制系统稳定性。在某些情况下，单个途径的损失导致补偿性 他人的贡献以维持系统地位。在其他情况下，部分不同的生物学途径部分 有助于计算单个功能，只有在损失或 淘汰所有个人贡献者。生物学的大规模实验和计算询问 为了建立系统级别的理解，需要组装。此类投资的范围 近年来，随着基于下一代的新多重工具的开发，近年来得到了极大的增强 测序（NGS）。这通常是通过单个细胞，单克隆抗体或其他细胞的标签来实现的 具有寡核苷酸条形码或唯一分子标识符（UMIS）的抗原粘合剂。这样的条形码/Umis可以 使用NGS独立遵循。此外，现在有可能开发基于CRISPR的淘汰图书馆 和针对整个基因组的筛选，或与某个功能相关的基因子集（例如细胞 糖基化）。该项目旨在通过解决该项目来结合NGS和CRISPR-CAS9的最新进展 假设'将CRISPR-CAS在大规模敲除基因的结合使用与NG相结合 测量相关的细胞系统响应可以以前所未有的规模告知我们系统属性。我们测试 使用糖基镜的例子的这种可能性。具体目的是：目标1。开发'凝集素标记 - Seq’，一种在复杂混合物中断言单个细胞的散热的方法。在这里，讲座和相关小组 mAb用独特的条形码寡核苷酸标记。这些试剂对 使用NGS大规模测量人类血液和乳房组织中的各种细胞类型。讲座结合 文献中现在可以使用大量碳水化合物结合蛋白的特异性，因此生物信息学 分析将应用于将单个细胞上的讲座结合测量与聚糖表位相关联和 单个细胞上存在的潜在碳氢结构。目标2。开发“ crispr-tag-seq” 同样，在单个单元格的水平上测得的SGRNA编辑给定的单元和相应的变化 多重凝集素/mAb结合曲线。在这里，针对调节细胞的347个基因的糖原-Crispr文库 将糖基化引入到乳腺癌细胞中。这种生化途径扰动的影响是 与讲座对单个细胞的结合有关。进行数学分析以分析SGRNA的效果 扰动。所有数据将在我们的网站VirtualGlyCome.org上向研究社区提供。长时间 运行，该项目中开发的方法可以扩展到其他细胞类型和生物学问题 最终简化并简化了我们对哺乳动物Glyce的理解。