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，几乎任何动物组织或胚胎都由数千个高度多样化的细胞组成。同样的基因组如何以及为什么导致细胞类型和功能如此巨大的多样性，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，几乎没有细胞特有的方法来联系因果关系。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实验性地解决这些瓶颈，这些方法和化学标记工具用于并行地对数千个单个细胞的表达、分类和图谱进行全基因组表征。这些信息被用来(I)实现对给定生物体内细胞类型的几乎完整的普查，重点放在对理解学习和记忆机制至关重要的动物模型上，如海兔，以及再生机制，如侧臂藻属，和(Ii)产生纳米级探针，选择性地标记特定细胞进行基因组编辑，而不考虑关于细胞分子多样性的任何高级知识。几个社区从拟议的研究中受益，包括比较神经生物学、发展、生物海洋学和新兴的合成生物学领域。该项目还为从本科生到博士后级别的受训人员提供跨学科培训机会，并针对不同的K-12学生群体开展海洋和比较生物学方面的教育推广活动。在我们理解基因组-物候组关系方面的重大挑战是，我们普遍无法在任何给定的位置和任何给定的时间在特定的单个细胞水平上操纵基因组操作。对于大多数无脊椎动物来说，当研究人员在几乎没有关于靶器官细胞组成的信息的情况下研究发育或神经元功能时，这些障碍更加戏剧性。在这里，用于大规模并行单细胞捕获和测序的微流控技术与新的细胞选择技术相结合，例如基于适体的细胞-SELEX，用于对完整组织中的单个细胞进行定量基因表达分析和成像。Aplysia(以及，一旦单细胞工具得到验证，侧臂藻和/或相关的鞭毛虫物种)被用来实现对其神经系统和效应器中的大多数细胞类型的几乎完整的全基因组分类。用于测量/控制已鉴定神经元中的基因表达的读出(S)是：具有归一化和绝对量化的靶基因表达水平的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，产生了各种细胞黏附分子和其他表面大分子结构的独特资源，这些结构对于设计和表征细胞特异性探针至关重要。然后，利用化学进化工具，对大规模生产细胞特异性适体/分子信标荧光探针的高通量系统进行了测试。最后，混合纳米级探针(例如，通过将细胞特异性荧光标记与核酸类似物偶联而成)被测试其将分子构建物自传递到靶细胞的能力，而无需直接注射、电穿孔或制造转基因动物。这个项目是由化学部生命过程化学项目共同资助的。

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.