Department of Bioengineering, Rice University, Houston, TX 77005, USA.
Department of BioSciences, Rice University, Houston, TX 77005, USA.
Nucleic Acids Res. 2022 Aug 12;50(14):7842-7855. doi: 10.1093/nar/gkac582.
Nuclease-inactivated CRISPR/Cas-based (dCas-based) systems have emerged as powerful technologies to synthetically reshape the human epigenome and gene expression. Despite the increasing adoption of these platforms, their relative potencies and mechanistic differences are incompletely characterized, particularly at human enhancer-promoter pairs. Here, we systematically compared the most widely adopted dCas9-based transcriptional activators, as well as an activator consisting of dCas9 fused to the catalytic core of the human CBP protein, at human enhancer-promoter pairs. We find that these platforms display variable relative expression levels in different human cell types and that their transactivation efficacies vary based upon the effector domain, effector recruitment architecture, targeted locus and cell type. We also show that each dCas9-based activator can induce the production of enhancer RNAs (eRNAs) and that this eRNA induction is positively correlated with downstream mRNA expression from a cognate promoter. Additionally, we use dCas9-based activators to demonstrate that an intrinsic transcriptional and epigenetic reciprocity can exist between human enhancers and promoters and that enhancer-mediated tracking and engagement of a downstream promoter can be synthetically driven by targeting dCas9-based transcriptional activators to an enhancer. Collectively, our study provides new insights into the enhancer-mediated control of human gene expression and the use of dCas9-based activators.
基于核酸酶失活的 CRISPR/Cas 系统（dCas 系统）已成为人为重塑人类表观基因组和基因表达的强大技术。尽管这些平台的应用越来越广泛，但它们的相对效力和机制差异尚未完全描述，特别是在人类增强子-启动子对中。在这里，我们系统比较了最广泛采用的基于 dCas9 的转录激活剂，以及由 dCas9 融合到人 CBP 蛋白催化核心组成的激活剂，在人类增强子-启动子对中。我们发现这些平台在不同的人类细胞类型中显示出不同的相对表达水平，并且它们的转录激活效率基于效应结构域、效应结构域募集结构、靶标基因座和细胞类型而变化。我们还表明，每个基于 dCas9 的激活剂都可以诱导增强子 RNA（eRNA）的产生，并且这种 eRNA 诱导与同源启动子下游 mRNA 的表达呈正相关。此外，我们使用基于 dCas9 的激活剂来证明人类增强子和启动子之间可以存在内在的转录和表观遗传相互作用，并且可以通过将基于 dCas9 的转录激活剂靶向增强子来人为地驱动增强子介导的下游启动子的跟踪和参与。总的来说，我们的研究为人类基因表达的增强子介导控制以及基于 dCas9 的激活剂的应用提供了新的见解。
