Inducible CRISPRa screen identifies putative enhancers

Zhongye Dai; Rui Li; Yuying Hou; Qian Li; Ke Zhao; Ting Li; Mulin Jun Li; Xudong Wu

doi:10.1016/j.jgg.2021.06.012

Inducible CRISPRa screen identifies putative enhancers

J Genet Genomics. 2021 Oct 20;48(10):917-927. doi: 10.1016/j.jgg.2021.06.012. Epub 2021 Jul 15.

Authors

Zhongye Dai¹, Rui Li¹, Yuying Hou¹, Qian Li¹, Ke Zhao², Ting Li¹, Mulin Jun Li², Xudong Wu³

Affiliations

¹ State Key Laboratory of Experimental Hematology, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Tianjin Key Laboratory of Cellular Homeostasis and Human Diseases, Department of Cell Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China.
² Department of Pharmacology, Tianjin Key Laboratory of Medical Epigenetics, Tianjin Medical University, Tianjin 300070, China.
³ State Key Laboratory of Experimental Hematology, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Tianjin Key Laboratory of Cellular Homeostasis and Human Diseases, Department of Cell Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China; Department of Neurosurgery, Tianjin Medical University General Hospital, Tianjin 300052, China; Tianjin Key Laboratory of Epigenetics for Organ Development of Premature Infants, Tianjin 300450, China. Electronic address: wuxudong@tmu.edu.cn.

PMID: 34531148
DOI: 10.1016/j.jgg.2021.06.012

Abstract

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.

Keywords: CRISPR activation; Cis-regulatory elements; Enhancer; Screen.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Clustered Regularly Interspaced Short Palindromic Repeats*