IOS EDGE: Nanoscale Probes and Infrastructure for Real-Time and Single-Cell Genomics across Metazoa

遗传物质或基因组首先在特定细胞水平上运作，实际上任何动物组织或胚胎都由数千个高度多样化的细胞组成。相同的基因组如何以及为什么会导致如此巨大的细胞类型和功能多样性，这是现代生物学尚未回答的问题。然而，对于大多数动物群体来说，连接因果关系的细胞特异性方法几乎不存在。这个跨学科的项目通过开发新的基因组方法和化学标记工具来解决这些瓶颈问题，这些方法和工具用于并行地对数千个单个细胞的表达、分类和映射进行全基因组表征。这些信息用于（i）实现给定生物体内细胞类型的几乎完整的普查，重点是对理解学习和记忆机制至关重要的动物模型，如Astrassia，以及再生，如Pleurobrachia，以及（ii）生成选择性标记特定细胞进行基因组编辑的纳米探针，无论有关细胞分子多样性的任何先进知识。几个社区正在受益于拟议的研究，包括比较神经生物学，发展，生物海洋学和合成生物学的新兴领域。 该项目还为从本科到博士后的学员提供跨学科培训机会，并为不同的K-12学生提供海洋和比较生物学方面的教育推广活动。我们理解基因组与表型组关系的最大挑战是，我们通常无法在任何给定位置和任何给定时间在特定个体细胞水平上操纵基因组操作。这些障碍对于大多数无脊椎动物来说更加引人注目，当研究人员研究发育或神经元功能时，关于靶器官的细胞组成的信息很少。在这里，用于大规模并行单细胞捕获和测序的微流体技术与新型细胞选择技术（如基于适体的Cell-SELEX）相结合，用于完整组织中单个细胞的定量基因表达分析和成像。Aaplasia（以及，一旦单细胞工具得到验证，侧臂和/或相关的栉水母物种）用于实现其神经系统和效应器官中大多数细胞类型的几乎完整的全基因组分类。用于测量/控制所鉴定的神经元中的基因表达的读数是：具有靶基因表达水平的标准化和绝对定量的scRNA-seq数据，以及q-RT-PCR。对照是靶基因不活跃或沉默的神经元。首先，产生了对设计和表征细胞特异性探针至关重要的多种细胞粘附分子和其他表面大分子结构的独特资源。然后，使用化学进化的工具，测试大规模制造细胞特异性适体/分子信标基荧光探针的高通量系统。最后，测试杂交纳米级探针（例如，通过将细胞特异性荧光标记物与核酸类似物偶联而制备）在不直接注射、电穿孔或不需要制备转基因动物的情况下将分子构建体自递送到靶细胞中的能力。该项目由化学系生命过程化学项目共同资助。

The genetic material, or genome, first and foremost operates at the level of specific cells, and practically any animal tissue or embryo consists of thousands of highly diverse cells. How and why the same genome leads to such enormous diversity of cell types and functions are unanswered questions of modern biology. Yet, cell-specific approaches to link cause and effect are virtually absent for a majority of animal groups. This interdisciplinary project addresses these bottlenecks experimentally by developing novel genomic approaches and chemical labeling tools for genome-wide characterization of expression, classification, and mapping of thousands of individual cells in parallel. This information is used to (i) achieve a nearly complete census of cell types within a given organism, focusing on animal models critical to understanding mechanisms of learning and memory, such as Aplysia, and regeneration, such as Pleurobrachia, and (ii) generate nanoscale probes that selectively mark specific cells for genome editing, regardless of any advance knowledge about the cells' molecular diversity. Several communities are benefiting from the proposed research, including comparative neurobiology, development, biological oceanography, and the emerging field of synthetic biology. The project also affords cross-disciplinary training opportunities for trainees from the undergraduate to postdoctoral level and educational outreach activities in marine and comparative biology aimed at a diverse K-12 student body. The grand challenge in our understanding of the genomes-to-phenomes relationships is our general inability to manipulate genome operation at the level of specific individual cells at any given location and at any given time. These obstacles are more dramatic for most invertebrates, when researchers study development or neuronal functions with little information about the cellular composition of target organs. Here, microfluidics for massive parallel single-cell capture and sequencing are integrated with novel cell selection technologies, such as aptamer-based-Cell-SELEX, for quantitative gene expression analyses and imaging of individual cells in intact tissues. Aplysia (and, once single-cell tools are validated, Pleurobrachia and/or related ctenophore species) are used to achieve nearly complete genome-wide classification of the majority of cell types in their neural systems and effector organs. The read-out(s) to measure/control gene expression in identified neurons are: scRNA-seq data with both normalized and absolute quantification of expression levels for target genes, and q-RT-PCR. Controls are neurons in which target genes are not active or silenced. First, unique resources for a diversity of cell adhesion molecules and other surface macromolecular structures critical to design and characterize cell-specific probes are generated. Then, using tools of chemical evolution, a high-throughput system to manufacture cell-specific aptamer-/molecular beacon-based fluorescent probes at a large scale is tested. Finally, hybrid nanoscale probes (e.g. made by coupling cell-specific fluorescent markers with nucleic acid analogues) are tested for their ability to self-deliver molecular constructs into target cells without direct injection, electroporation, or the need to make transgenic animals. This project is co-funded by the Chemistry of Life Processes program in the Division of Chemistry.