Multiplexed Engineering and Analysis of Combinatorial Enhancer Activity in Single Cells.
Shiqi Xie, Jialei Duan, Boxun Li, Pei Zhou, Gary C Hon
Author Information
- Shiqi Xie: Laboratory of Regulatory Genomics, Cecil H. and Ida Green Center for Reproductive Biology Sciences, Division of Basic Reproductive Biology Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
- Jialei Duan: Laboratory of Regulatory Genomics, Cecil H. and Ida Green Center for Reproductive Biology Sciences, Division of Basic Reproductive Biology Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
- Boxun Li: Laboratory of Regulatory Genomics, Cecil H. and Ida Green Center for Reproductive Biology Sciences, Division of Basic Reproductive Biology Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
- Pei Zhou: Laboratory of Regulatory Genomics, Cecil H. and Ida Green Center for Reproductive Biology Sciences, Division of Basic Reproductive Biology Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
- Gary C Hon: Laboratory of Regulatory Genomics, Cecil H. and Ida Green Center for Reproductive Biology Sciences, Division of Basic Reproductive Biology Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. Electronic address: gary.hon@utsouthwestern.edu.
The study of enhancers has been hampered by the scarcity of methods to systematically quantify their endogenous activity. We develop Mosaic-seq to systematically perturb enhancers and measure their endogenous activities at single-cell resolution. Mosaic-seq uses a CRISPR barcoding system to jointly measure a cell's transcriptome and its sgRNA modulators, thus quantifying the effects of dCas9-KRAB-mediated enhancer repression in single cells. Applying Mosaic-seq to 71 constituent enhancers from 15 super-enhancers, our analysis of 51,448 sgRNA-induced transcriptomes finds that only a small number of constituents are major effectors of target gene expression. Binding of p300 and RNAPII are key features of these constituents. We determine two key parameters of enhancer activity in single cells: their penetrance in a population and their contribution to expression in these cells. Through combinatorial interrogation, we find that simultaneous repression of multiple weak constituents can alter super-enhancer activity in a manner greatly exceeding repression of individual constituents.
CRISPR-Cas Systems
Cell Separation
Databases, Genetic
Enhancer Elements, Genetic
Flow Cytometry
Gene Expression Profiling
Gene Expression Regulation
Genotype
HEK293 Cells
High-Throughput Nucleotide Sequencing
Humans
K562 Cells
Penetrance
Phenotype
RNA Polymerase II
RNA, Guide, CRISPR-Cas Systems
Single-Cell Analysis
Transcription Factors
Transcription, Genetic
Transcriptional Activation
Transcriptome
Transfection
Transposases
p300-CBP Transcription Factors
RNA, Guide, CRISPR-Cas Systems
Tn5 transposase
Transcription Factors
p300-CBP Transcription Factors
RNA Polymerase II
Transposases
