Single cell tagging of localized RNA from whole populations

来自整个群体的局部 RNA 的单细胞标记

基本信息

批准号：
10096934
负责人：
GARY D STORMO
金额：
$ 33.86万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-20 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10096934
关键词：
Affinity Animal Model Animals Bar Codes Binding Binding Proteins Biotechnology CRISPR interference CRISPR library Cataloging Catalogs Cell Culture Techniques Cell Separation Cells Clustered Regularly Interspaced Short Palindromic Repeats Code Complex Custom Cytoplasmic Granules DNA DNA Sequence Data Data Pooling Development Elements Estrogen receptor positive Gene Expression Gene Expression Regulation Genes Genetic Genetic Diseases Genetic Screening Genetic Variation Genetic study Genome Goals Guide RNA Individual Invertebrates Label Lentivirus Vector Ligation Link Location Mammalian Cell Mammalian Genetics Measures Messenger RNA Methods Mitochondria Poly U Poly(A) Tail Population Proteins RNA RNA analysis RNA-Binding Proteins RNA-Directed DNA Polymerase Regulatory Element Reporter Genes Resolution Ribonucleases Ribosomal Proteins Ribosomes Sampling Specificity Technology Tissues Transcript Untranslated RNA Viral Vector Work aptamer base design disease phenotype experimental study gene function gene repression genetic variant genome editing human disease in vivo knock-down mRNA tagging mammalian genome new technology single cell analysis single cell sequencing single cell technology small hairpin RNA transcriptome

项目摘要

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.

项目摘要/摘要该提案的目的是开发一种广泛适用的技术，称为Scalop（单个单元格）分析整个群体上的局部RNA），以测量单个细胞中的基因表达，而没有需要对单个细胞进行技术挑战。目前，大规模遗传基于新技术的屏幕导致了我们对哺乳动物的理解的重要进展基因组和与人类疾病有关的遗传变异。但是，这些屏幕的功能受到缺乏测量这些大筛选中基因表达的方法。我们提出的技术将填补这一点主要的未满足需求，因此对哺乳动物遗传学产生了广泛的影响。我们的目标是开发一种方法使用合并样品中的RNA接近连接，将单细胞特异性序列条形码附加到转录本上。我们建议通过设计条形码的“ tagrnas”来做到这一点，该条形码在单元中表达并针对特定 RNA结合蛋白。这些tagrnas附着在从同一单个单元中得出的转录本接近连接。为了开发这种方法，我们的目标是1）优化和量化方法的效率链接tagrnas和细胞RNA，2）优化体内特异性和单细胞分辨率的Scaleop，3）为天然蛋白质和替代位置靶标开发RNA适体。