@article{b116db79c3c84a4780a2f200382a154b,
title = "Multiplexed Engineering and Analysis of Combinatorial Enhancer Activity in Single Cells",
abstract = "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",
author = "Shiqi Xie and Jialei Duan and Boxun Li and Pei Zhou and Hon, {Gary C.}",
note = "Funding Information: We gratefully acknowledge Steven McCarroll for extensive protocols on Drop-seq. We also thank Charles Gersbach for his gift of the pLV hU6-sgRNA-hUbC-dCas9-KRAB-T2a-Puro construct, Rickard Sandberg for his gift of Tn5 transposase plasmid, W. Lee Kraus for critical reading of this manuscript, and the journal reviewers whose comments have significantly improved this manuscript. This work is supported by the Cancer Prevention Research Institute of Texas (CPRIT) grant RR140023 (to G.C.H.) and an American Heart Association fellowship 16POST29910007 (to S.X.). The authors acknowledge the BioHPC computational infrastructure at UT Southwestern as well as the Texas Advanced Computing Center (TACC) at The University of Texas at Austin for providing HPC and storage resources that have contributed to the research results reported within this paper (https://www.tacc.utexas.edu). The authors also acknowledge UT Southwestern's McDermott Center and the Children's Research Institute for providing next-generation sequencing services for this work. Publisher Copyright: {\textcopyright} 2017 Elsevier Inc.",
year = "2017",
month = apr,
day = "20",
doi = "10.1016/j.molcel.2017.03.007",
language = "English (US)",
volume = "66",
pages = "285--299.e5",
journal = "Molecular cell",
issn = "1097-2765",
publisher = "Cell Press",
number = "2",
}