Rui Li
Yuying Hou
Qian Li
Ke Zhao
Ting Li
Mulin Jun Li
Xudong Wu
a. 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;
b. Department of Pharmacology, Tianjin Key Laboratory of Medical Epigenetics, Tianjin Medical University, Tianjin 300070, China;
c. Department of Neurosurgery, Tianjin Medical University General Hospital, Tianjin 300052, China;
d. Tianjin Key Laboratory of Epigenetics for Organ Development of Premature Infants, Tianjin 300450, China
Funds: 2018KJ075 to T.L.), Open grant from the Chinese Academy of Medical Sciences (157-Z20-04 to X.W.). We also appreciate the support from Tianjin Postgraduate Research Innovation Project (2019YJSB117 to Z.D.
2019YJSS176 to Y.H.).
31900464 to T.L.), and the Natural Science Foundation of Tianjin Municipal Science and Technology Commission (18JCJQJC48200 to X.W.), Tianjin Education Commission (2020ZD13 to X.W.
We thank members of the Wu laboratory for discussions. This work was supported by the National key research and development program (2017YFA0504102 to X.W.), the National Natural Science Foundation of China (81772676, 31970579 to X.W.
Received Date: 2021-03-11
Accepted Date:2021-06-07
Rev Recd Date:2021-05-21
Publish Date:2021-07-15
Abstract
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
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