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.

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，在天然细胞环境中鉴定增强剂仍然是一个挑战。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。有了这条线，简单地引入靶向启动子或增强子的特定引导 RNA 就可以让我们以可诱导、可逆和 Dox 浓度依赖性方式实现 CRISPRa 的作用。利用这个系统，我们在T ( Brachyury)，关键的中胚层发育调节基因之一。此外，我们确定了几个具有假定增强子染色质特征的 CRISPRa 响应元件，包括人类具有身高风险变异的同源序列区域。ESC 中该区域的遗传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 系列为推定增强剂的高通量筛选提供了一个方便的平台。