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

IOS EDGE：用于后生动物实时和单细胞基因组学的纳米级探针和基础设施

基本信息

批准号：
1645219
负责人：
Leonid Moroz
金额：
$ 100万
依托单位：
University of Florida
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2017
资助国家：
美国
起止时间：
2017-07-15 至 2021-06-30
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1645219&HistoricalAwards=false
关键词：
IOS EDGE Nanoscale Probes Infrastructure

项目摘要

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.

遗传物质或基因组首先在特定细胞的水平上运行，实际上，任何动物组织或胚胎都由数千种高度多样化的细胞组成。相同的基因组如何以及为什么导致细胞类型和功能的多样性是现代生物学的未解决问题。然而，对于大多数动物群体而言，几乎没有细胞特异性的联系因果关系。该跨学科项目通过开发新型的基因组方法和化学标记工具来解决这些瓶颈，以平行地表达，分类和成千上万个单个细胞的绘制。这些信息用于（i）在给定有机体中实现细胞类型几乎完整的普查，重点是了解学习和记忆机制的至关重要的动物模型，例如丙酶和再生，例如胸膜炎和再生，以及（ii）生成纳米级探针，这些纳米级探针选择性地标记了具有基因组编辑的特定细胞，无论对细胞的知识有任何提前的知识，都可以选择特定的细胞。几个社区受益于拟议的研究，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。该项目还为学员提供了跨学科培训机会，从本科到博士后水平以及海洋和比较生物学的教育外展活动，针对不同的K-12学生团体。我们对基因组与表现组关系的理解的巨大挑战是，我们通常无法在任何给定位置和任何给定时间操纵特定单个细胞的基因组操作。对于大多数无脊椎动物而言，这些障碍更为戏剧性，当研究人员研究开发或神经元功能，几乎没有有关靶器官细胞组成的信息。在这里，用于大规模平行单细胞捕获和测序的微流体与新颖的细胞选择技术（例如基于适体基细胞 - 丝体）集成，用于定量基因表达分析和对完整组织中单个细胞的成像。 Aplysia（以及一旦验证了单细胞工具，Pleurobrachia和/或相关的Cenophore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类型分类。测量已识别神经元中测量/控制基因表达的读出是：靶基因表达水平的归一化和绝对定量的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，生成了针对设计和表征细胞特异性探针至关重要的其他表面大分子结构的独特资源。然后，测试了使用化学演化工具，一种高通量系统来制造大规模的细胞特异性适体/分子信标的荧光探针。最后，测试了杂化纳米级探针（例如，通过耦合细胞特异性荧光标记与核酸类似物制成），以实现其在不直接注射，电孔或进行转基因动物的情况下将分子构建到靶细胞中的能力。该项目由化学过程的化学过程中的化学计划共同资助。

项目成果

期刊论文数量（30）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Glycine as a signaling molecule and chemoattractant in Trichoplax (Placozoa): insights into the early evolution of neurotransmitters

甘氨酸作为毛盘菌（Placozoa）中的信号分子和化学引诱剂：深入了解神经递质的早期进化

DOI：
10.1097/wnr.0000000000001436
发表时间：
2020
期刊：
NeuroReport
影响因子：
1.7
作者：
Romanova, Daria Y.;Heyland, Andreas;Sohn, Dosung;Kohn, Andrea B.;Fasshauer, Dirk;Varoqueaux, Frederique;Moroz, Leonid L.
通讯作者：
Moroz, Leonid L.

Comparative neuroanatomy of ctenophores: Neural and muscular systems in Euplokamis dunlapae and related species

DOI：
10.1002/cne.24770
发表时间：
2019-10-04
期刊：
JOURNAL OF COMPARATIVE NEUROLOGY
影响因子：
2.5
作者：
Norekian, Tigran P.;Moroz, Leonid L.
通讯作者：
Moroz, Leonid L.

Life Strategies in Placozoa

Placozoa 的生活策略

DOI：
10.1101/2021.11.26.470175
发表时间：
2021
期刊：
bioRxiv
影响因子：
0
作者：
Romanova, D.Y.;Mikhail A. Nikitin, M.A.;Sergey V. Shchenkov, S.V.;Moroz, L.L.
通讯作者：
Moroz, L.L.

The diversification and lineage-specific expansion of nitric oxide signaling in Placozoa: insights in the evolution of gaseous transmission

DOI：
10.1038/s41598-020-69851-w
发表时间：
2020-08-03
期刊：
SCIENTIFIC REPORTS
影响因子：
4.6
作者：
Moroz，Leonid L.;Romanova，Daria Y.;Fasshauer，Dirk
通讯作者：
Fasshauer，Dirk

Enhanced Targeted Gene Transduction: AAV2 Vectors Conjugated to Multiple Aptamers via Reducible Disulfide Linkages.

DOI：
10.1021/jacs.7b08518
发表时间：
2018-01-10
期刊：
Journal of the American Chemical Society
影响因子：
15
作者：
Wu Y;Zhang L;Cui C;Cansiz S;Liang H;Wu C;Teng IT;Chen W;Liu Y;Hou W;Zhang X;Tan W
通讯作者：
Tan W