Single cell tagging of localized RNA from whole populations

项目摘要/摘要 这项提议的目标是开发一种广泛适用的技术，称为SCALoP(Single Cell 分析整个种群的定位RNA)，以测量单个细胞中的基因表达，而不是 需要在单个细胞上执行具有技术挑战性的操作。目前，大规模的基因工程 基于新技术的屏幕正在引领我们对哺乳动物的理解取得重大进展 与人类疾病有关的基因组和遗传变异。然而，这些屏幕的功率受到 缺乏在这些大屏幕上测量基因表达的方法。我们提出的技术将填补这一空白 主要的未得到满足的需求，因此对哺乳动物的遗传学有广泛的影响。我们的目标是开发一种方法来 在混合样本中使用RNA邻近连接将单细胞特定序列条形码连接到转录本上。 我们建议通过设计条形码“tag RNAs”来实现这一点，这种“tag RNAs”在细胞中表达，并针对特定的 RNA结合蛋白。这些标签RNA通过以下方式连接到来自同一单个细胞的转录本 近距离结扎。为了发展这种方法，我们的目标是1)优化和量化方法的效率，以 链接标签RNA和细胞RNA；2)优化SCALoP的体内特异性和单细胞分辨率；3) 开发针对天然蛋白质和交替定位靶标的RNA适配子。

Project Summary/Abstract The objective of this proposal is to develop a broadly applicable technology, called SCALoP (Single Cell Analysis of Localized RNA on whole Populations), to measure gene expression in single cells, without the need to perform technically challenging manipulations on individual cells. Currently, large-scale genetic screens based on new technologies are leading to important advances in our understanding of mammalian genomes and genetic variation linked to human disease. However, the power of these screens is limited by the lack of methods to measure gene expression in these large screens. Our proposed technology would fill this major unmet need, and thus have a broad impact on mammalian genetics. Our goal is to develop a method to attach single-cell-specific sequence barcodes to transcripts, using RNA proximity ligation in pooled samples. We propose to do this by designing barcoded “tagRNAs” which are expressed in cells and targeted to specific RNA binding proteins. These tagRNAs are attached to transcripts derived from the same single cell by proximity ligation. To develop this method, our aims are 1) optimize and quantify the efficiency of methods to link tagRNAs and cellular RNA, 2) optimize in vivo specificity and single cell resolution of SCALoP, and 3) develop RNA aptamers to natural proteins and alternate location targets.