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

• 批准号: 10266095
• 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
• 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/10266095
• 关键词: Affinity; Animal Model; Animals; Bar Codes; Binding; Binding Proteins; Biotechnology; CRISPR interference; CRISPR library; Cataloging; 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; 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

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.

项目总结/摘要 该提案的目的是开发一种广泛适用的技术，称为SCALoP（单细胞 在整个群体上的局部RNA的分析），以测量单细胞中的基因表达，而不需要在单个细胞中进行局部RNA的分析。 需要对单个细胞进行技术上具有挑战性的操作。目前，大规模基因 基于新技术的屏幕在我们对哺乳动物的理解方面取得了重要进展 与人类疾病有关的基因组和遗传变异。然而，这些屏幕的功能受到 缺乏在这些大屏幕中测量基因表达的方法。我们提出的技术将填补这一空白 主要未满足的需求，从而对哺乳动物遗传学产生广泛影响。我们的目标是开发一种方法， 在合并的样品中使用RNA邻近连接将单细胞特异性序列条形码附接至转录物。 我们建议通过设计在细胞中表达并靶向特异性的条形码“tagRNA”来实现这一点。 RNA结合蛋白。这些tagRNA通过以下方式附着于来自同一单细胞的转录物： 邻位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， 连接tagRNA和细胞RNA，2）优化SCALoP的体内特异性和单细胞分辨率，以及3） 开发天然蛋白质和替代定位靶标的RNA适体。