High content glycomics analysis using next generation sequencing technology

生物系统通常很强大，因为有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，单一途径的丧失导致代偿性的 其他人的捐款，以维持系统地位。在其他情况下，许多不同的生物途径部分 有助于调节单一功能，只有在功能丧失时才能观察到完全功能丧失， 淘汰所有个人贡献者。大规模的生物学实验和计算研究 汇编是必要的，以便发展系统级的理解。此类调查的范围 近年来，随着基于下一代的新型多路复用工具的开发， 测序（NGS）。这通常通过标记单个细胞、单克隆抗体或其他生物标记来实现。 具有寡核苷酸条形码或独特分子标识符（UMI）的抗原结合物。这种条形码/UMI可以 使用NGS独立跟踪。此外，现在可以开发基于CRISPR的敲除文库， 并筛选靶向整个基因组或与特定功能（例如细胞功能）相关的基因子集 糖基化）。该项目旨在通过解决以下问题，将NGS和CRISPR-Cas9的最新进展联合收割机结合起来： 假设“CRISPR-Cas大规模敲除基因与NGS结合使用， 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特性。我们测试 这种可能性使用的例子从糖科学。具体目标是：目标1。为了开发'凝集素-标签- SEQ“，一种测定复杂混合物中单个细胞的糖组的方法。在这里，一组凝集素和相关的 mAb用独特条形码化的寡核苷酸标记。这些试剂同时结合到 使用NGS大规模测量人类血液和乳腺组织中的多种细胞类型。凝集素结合 大量碳水化合物结合蛋白的特异性现在可在文献中获得，因此生物信息学 将应用分析以将单个细胞上的凝集素结合测量与聚糖表位相关联， 存在于单个细胞上的潜在碳水化合物结构。目标2.开发“CRISPR-Tag-seq”， 在单个细胞水平上同时测量编辑给定细胞的sgRNA和细胞内的相应变化。 多重凝集素/mAb结合谱。在这里，一个靶向347个调控细胞增殖的基因的糖原-CRISPR文库， 糖基化被引入乳腺癌细胞。这种生物化学途径扰动的影响是 与单个细胞上的凝集素结合有关。进行数学分析以分析sgRNA的作用。 扰动所有数据都将在我们的网站VirtualGlycome.org上提供给研究社区。 运行，在这个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， 最终简化我们对哺乳动物糖组的理解。

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.