Inducible CRISPRa screen identifies putative enhancers
Inducible CRISPRa screen identifies putative enhancers
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诱导型 CRISPRa 筛选鉴定出假定的增强子
DOI：
10.1016/j.jgg.2021.06.012
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发表时间：
2021-10-20
影响因子：
5.9
通讯作者：
Wu, Xudong
中科院分区：
文献类型：
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作者：
Dai, Zhongye;Li, Rui;Wu, Xudong
增强子是调节时空基因表达和控制细胞命运的关键顺式调节元件。然而，在天然细胞环境中识别增强子仍然是一个挑战。在这里，我们通过在小鼠胚胎干细胞 (iVPR ESC) 中转基因表达多西环素 (Dox) 诱导型 dCas9-VPR 来开发诱导型 CRISPR 激活 (CRISPRa) 系统。通过该产品线，简单引入靶向启动子或增强子的特异性向导RNA，使我们能够以可诱导、可逆和Dox浓度依赖性方式实现CRISPRa的效果。利用该系统，我们在 T (Brachyury)（关键的中胚层发育调节基因之一）周围的基因组区域（105 kilobase）诱导平铺 CRISPRa。此外，我们还鉴定了几个具有假定增强子染色质特征的 CRISPRa 响应元件，其中包括人类携带身高风险变异的同源序列区域。 ESC 中该区域的基因缺失确实会影响随后的 T 基因激活和成骨分化。因此，我们的诱导型 CRISPRa ESC 系为推定增强子的高通量筛选提供了便利的平台。版权所有(C)2021，中国科学院遗传与发育生物学研究所，中国遗传学会。由爱思唯尔有限公司和科学出版社出版。版权所有。
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.