Mountain View
biomedical and healthCAre Data Discovery Index Ecosystem
help Advanced Search
Title: Multiplexed engineering and analysis of endogenous enhancer activity in single cells: Mosaic-Seq of beta-globin locus (separate infection)      
keywords:
Transcriptome or Gene expression
ID:
PRJNA322846
description:
Transcriptional enhancers orchestrate cell-type specific gene expression programs critical to eukaryotic development, physiology, and disease. However, despite the large number of enhancers now identified, only a small number have been functionally assessed. Here, we develop MOsaic Single-cell Analysis by Indexed CRISPR Sequencing (Mosaic-seq), a method that measures one direct phenotype of enhancer repression: change of the transcriptome, at the single cell level. Using dCas9-KRAB to suppress enhancer function, we first implement a multiplexed system to allow the simultaneous measurement of the transcriptome and detection of sgRNAs by single cell RNA sequencing. We validate this approach by targeting the HS2 enhancer in the well-studied beta-globin locus. Next, through computational simulation, we demonstrate strategies to robustly detect changes in gene expression in these single cell measurements. Finally, we use Mosaic-seq to target 71 hypersensitive regions belonging to 15 super-enhancers in K562 cells by utilizing a lentiviral library containing 241 unique-barcoded sgRNAs. Our results demonstrate that Mosaic-seq is a reliable approach to study enhancer function in single cells in a high-throughput manner. Overall design: The overall design consists of RNA sequencing in bulk and in single K562 cells. As a control, we performed single-cell RNA sequencing of K562 cells expressing dCas9-KRAB and a control sgRNA targeting the non-expressed HSBP1 gene in two biological replicates. As a pilot project, we performed single cell RNA-Seq of dCas9-KRAB K562 cells after pooled infection of a library of 10 sgRNA viruses targeting promoters and enhancers in the beta-globin locus. As controls for this experiment, we individually infected these 10 sgRNAs and either performed 1) bulk RNA sequencing or 2) single cell RNA sequencing after pooling the separately infected cells. Finally, we scaled this single-cell approach to measure transcriptome changes for a library of 241 sgRNAs spanning 71 hypersensitive sites from 15 super-enhancers in K562 cells.
accesstypes:
download
landingpage: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA322846
authentication:
none
authorization:
none
ID:
pmid:28416141
name:
Homo sapiens
ncbiID:
ncbitax:9606
abbreviation:
NCBI
homePage: http://www.ncbi.nlm.nih.gov
ID:
SCR:006472
name:
National Center for Biotechnology Information
homePage: http://www.ncbi.nlm.nih.gov/bioproject
ID:
SCR:004801
name:
NCBI BioProject