Multiplexed Engineering and Analysis of Combinatorial Enhancer Activity in Single Cells

Mol Cell. 2017 Apr 20;66(2):285-299.e5. doi: 10.1016/j.molcel.2017.03.007. Epub 2017 Apr 13.


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.

Keywords: CRISPR; enhancers; epigenetics; gene editing; gene regulation; single-cell RNA-seq.

MeSH terms

  • CRISPR-Cas Systems
  • Cell Separation / methods
  • Databases, Genetic
  • Enhancer Elements, Genetic*
  • Flow Cytometry
  • Gene Expression Profiling / methods*
  • Gene Expression Regulation
  • Genotype
  • HEK293 Cells
  • High-Throughput Nucleotide Sequencing*
  • Humans
  • K562 Cells
  • Penetrance
  • Phenotype
  • RNA Polymerase II / genetics
  • RNA Polymerase II / metabolism
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • RNA, Guide, CRISPR-Cas Systems / metabolism
  • Single-Cell Analysis / methods*
  • Transcription Factors / genetics*
  • Transcription Factors / metabolism
  • Transcription, Genetic*
  • Transcriptional Activation*
  • Transcriptome*
  • Transfection
  • Transposases / genetics
  • Transposases / metabolism
  • p300-CBP Transcription Factors / genetics
  • p300-CBP Transcription Factors / metabolism


  • RNA, Guide, CRISPR-Cas Systems
  • Tn5 transposase
  • Transcription Factors
  • p300-CBP Transcription Factors
  • RNA Polymerase II
  • Transposases