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。相同的基因组如何以及为何导致如此巨大的细胞类型和功能多样性是现代生物学尚未解答的问题。然而，对于大多数动物群体来说，实际上不存在连接因果关系的细胞特异性方法。这个跨学科项目通过开发新颖的基因组方法和化学标记工具，对数千个单个细胞的表达、分类和绘图进行全基因组表征，通过实验解决这些瓶颈。这些信息用于 (i) 对给定生物体内的细胞类型进行近乎完整的普查，重点关注对理解学习和记忆机制至关重要的动物模型，例如海兔和再生机制，例如侧腕动物，以及 (ii) 生成纳米级探针可以选择性地标记特定细胞以进行基因组编辑，而不管细胞分子多样性的先进知识如何。一些社区正在从拟议的研究中受益，包括比较神经生物学、发育、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水平的学员提供跨学科培训机会，并为多样化的 K-12 学生群体提供海洋和比较生物学的教育外展活动。我们理解基因组与现象关系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在特定个体细胞的水平上操纵基因组操作。对于大多数无脊椎动物来说，当研究人员研究发育或神经元功能时，有关目标器官细胞组成的信息很少，这些障碍更为严重。在这里，用于大规模并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。海兔（以及一旦单细胞工具得到验证，侧腕动物和/或相关栉水母物种）可用于对其神经系统和效应器官中的大多数细胞类型实现近乎完整的全基因组分类。用于测量/控制已识别神经元中基因表达的读数包括：具有目标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。对照是目标基因不活跃或沉默的神经元。首先，生成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，使用化学进化工具，测试大规模制造细胞特异性适体/分子信标荧光探针的高通量系统。最后，测试混合纳米级探针（例如通过将细胞特异性荧光标记与核酸类似物偶联而制成）将分子构建体自我递送到靶细胞中的能力，而无需直接注射、电穿孔或不需要制备转基因动物。该项目由化学系生命过程化学项目共同资助。

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）

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.

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.

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