Systematic comparison of CRISPR-based transcriptional activators uncovers gene-regulatory features of enhancer-promoter interactions.
Systematic comparison of CRISPR-based transcriptional activators uncovers gene-regulatory features of enhancer-promoter interactions.
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DOI：
10.1093/nar/gkac582
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发表时间：
2022-08-12
影响因子：
14.9
通讯作者：
Hilton, Isaac B.
中科院分区：
文献类型：
--
作者：
Wang, Kaiyuan;Escobar, Mario;Li, Jing;Mahata, Barun;Goell, Jacob;Shah, Spencer;Cluck, Madeleine;Hilton, Isaac B.
基于核酸酶失活的 CRISPR/Cas（基于 dCas）系统已成为综合重塑人类表观基因组和基因表达的强大技术。尽管这些平台越来越多地被采用，但它们的相对效力和机制差异尚未完全表征，特别是在人类增强子-启动子对方面。在这里，我们在人类增强子-启动子对上系统地比较了最广泛采用的基于 dCas9 的转录激活子，以及由融合到人类 CBP 蛋白催化核心的 dCas9 组成的激活子。我们发现这些平台在不同的人类细胞类型中表现出不同的相对表达水平，并且它们的反式激活功效根据效应器结构域、效应器招募架构、目标位点和细胞类型而变化。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 诱导与同源启动子的下游 mRNA 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互惠性，并且增强子介导的下游启动子的跟踪和接合可以通过将基于 dCas9 的转录激活剂靶向增强子来综合驱动。总的来说，我们的研究为增强子介导的人类基因表达控制和基于 dCas9 的激活剂的使用提供了新的见解。
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.
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