Development of CLADE: Cell Lineage Annotating DNA Elements

摘要 基于液滴的单细胞RNA测序(scRNA-seq)的最新进展 技术已经改变了几个研究领域的格局。它已经启用了 研究人员试图阐明组织、器官和肿瘤中的细胞复杂性 传统上被视为同质。新的视角对于理解 正常和疾病条件下的生物学，它将刺激新的 利用对生理系统更细粒度的了解的疗法。 随着这个新视角的出现，人们意识到了scrna-seq的一些局限性和障碍。 必须克服这一点，才能实现单细胞分析的潜力。这项提案的重点是 开发一种创新的新技术，将显著提高scRNA的影响- SEQ技术应用于干细胞研究和再生医学疗法。目前， Scrna-seq可以很好地识别样本中的细胞成分，但它不能 辨别单个细胞通过细胞谱系的路线。确定细胞是至关重要的 谱系，因为它们提供了通知定向协议的基本指令 分化和基于细胞的治疗。该提案的系统名为细胞谱系注解 DNA元件(Clade)将把一个单独条形码的质粒库引入干细胞 DNA。利用来自Epstein-Barr病毒的上体DNA维持系统的元件， 当细胞经历细胞分裂时，分支质粒条形码将发生变化，从而产生一个独特的队列 单个细胞的条形码质粒组。通过使用相同的反应产生scRNA-seq 抄本和支系识别，系统将提供一种无缝的方式 根据细胞的转录特征来确定细胞的祖先。通过一系列的六个 作为里程碑，将对Clade系统进行合成、测试和开发，以便能够 使用scRNA-seq和干细胞的其他组织很容易使用。我们预计Clade将使 将为&gt；10的40,000个单个细胞确定近乎全面的细胞祖先 几代人的细胞分裂。与目前数百个电池和两个或以上的电池相比 三次细胞分裂，Clade可以极大地增加识别细胞谱系和 对受益于scRNA-seq研究的领域产生长期持久的影响。

ABSTRACT The relatively recent development of droplet-based single cell RNA sequencing (scRNA-seq) techniques has changed the landscape of several fields of research. It has enabled investigators to elucidate the cellular complexities in tissues, organs, and tumors which have traditionally been treated as homogeneous. The new perspective is important for understanding the biology underlying normal and disease conditions, and it will stimulate development of new therapies that take advantage of the more granular understanding of physiological systems. With this new perspective comes a realization of some of the limits to scRNA-seq, and barriers that must be overcome to achieve potential of single cell analyses. This proposal focuses on developing an innovative new technology that will dramatically improve the impact of scRNA- seq techniques applied to stem cell research and regenerative medicine therapies. Currently, scRNA-seq is great at identifying the cellular components within a sample, but it is incapable of discerning the route individual cells took through a cell lineage. It is critical to determine cell lineages because they provide the essential instructions informing protocols for directed differentiation and cell based therapies. The proposal's system, named Cell Lineage Annotating DNA Elements (CLADE) will introduce into stem cells a library of individually barcoded plasmid DNAs. Using elements of episomal DNA maintenance system from the Epstein-Barr virus, the CLADE plasmid barcode will change as cells undergo cell divisions, resulting in a unique cohort of barcoded plasmids for individual cells. By using the same reaction to generate scRNA-seq transcriptomes and CLADE identification, the system will provide a seamless way of determining ancestry of cells alongside their transcriptomic identity. Through a series of six Milestones, the CLADE system will be synthesized, tested, and developed so that it can be readily used by other groups using scRNA-seq and stem cells. We anticipate CLADE will enable a near comprehensive cellular ancestry to be determined for >40,000 individual cells for >10 generations of cell division. Compared to current capabilities of hundreds of cells and two or three cell divisions, CLADE can massively increase capacity for identifying cell lineages and have a long lasting impact on fields that benefit from scRNA-seq research.