Inducible CRISPRa screen identifies putative enhancers

被引：18

作者：

Dai, Zhongye ^{[1
]}

Li, Rui ^{[1
]}

Hou, Yuying ^{[1
]}

Li, Qian ^{[1
]}

Zhao, Ke ^{[2
]}

Li, Ting ^{[1
]}

Li, Mulin Jun ^{[2
]}

Wu, Xudong ^{[1
,3
,4
]}

机构：

[1] Tianjin Med Univ, Sch Basic Med Sci,Dept Cell Biol, Collaborat Innovat Ctr Med Epigenet,State Key Lab, Tianjin Key Lab Cellular Homeostasis & Human Dis, Tianjin 300070, Peoples R China

[2] Tianjin Med Univ, Dept Pharmacol, Tianjin Key Lab Med Epigenet, Tianjin 300070, Peoples R China

[3] Tianjin Med Univ Gen Hosp, Dept Neurosurg, Tianjin 300052, Peoples R China

[4] Tianjin Key Lab Epigenet Organ Dev Premature Infa, Tianjin 300450, Peoples R China

来源：

JOURNAL OF GENETICS AND GENOMICS | 2021年 / 48卷 / 10期

基金：

中国国家自然科学基金;

关键词：

CRISPR activation; Cis-regulatory elements; Enhancer; Screen; EMBRYONIC STEM; GENE; EXPRESSION; NAIVE; INTERROGATION; ACTIVATION; SYSTEM; CELLS;

D O I：

10.1016/j.jgg.2021.06.012

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Enhancers are critical cis-regulatory elements that regulate spatiotemporal gene expression and control cell fates. However, the identification of enhancers in native cellular contexts still remains a challenge. Here, we develop an inducible CRISPR activation (CRISPRa) system by transgenic expression of doxycycline (Dox)inducible dCas9-VPR in mouse embryonic stem cells (iVPR ESC). With this line, a simple introduction of specific guide RNAs targeting promoters or enhancers allows us to realize the effect of CRISPRa in an inducible, reversible, and Dox concentration-dependent manner. Taking advantage of this system, we induce tiled CRISPRa across genomic regions (105 kilobases) surrounding T (Brachyury), one of the key mesodermal development regulator genes. Moreover, we identify several CRISPRa-responsive elements with chromatin features of putative enhancers, including a region the homologous sequence in which humans harbors a body height risk variant. Genetic deletion of this region in ESC does affect subsequent T gene activation and osteogenic differentiation. Therefore, our inducible CRISPRa ESC line provides a convenient platform for high-throughput screens of putative enhancers. Copyright (C) 2021, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.

引用

页码：917 / 927

页数：11

共 42 条

[1] Genome-Wide Quantitative Enhancer Activity Maps Identified by STARR-seq [J].