Department of BioSciences, Rice University, Houston, TX, USA.
Department of Bioengineering, Rice University, Houston, TX, USA.
Methods Mol Biol. 2022;2518:203-215. doi: 10.1007/978-1-0716-2421-0_12.
CRISPR-Cas regulators have provided an excellent toolbox to control gene expression due to the versatility of its components and the easy programming of the single guide RNA (sgRNA) to target DNA sequences. Included in this are CRISPR activation (CRISPRa) systems. These systems allow users to activate transcription of a target gene through the localization of transcription activation domains (ADs) near promoter elements, which in turn recruit RNA polymerase (RNAP) to turn on transcription. A variety of different CRISPRa systems have been described that vary in AD type, recruitment strategies, and CRISPR-Cas systems. Recently, a highly modular CRISPRa system was described that allows for facile exchange of ADs and CRISPR-Cas components. This allows for the creation of CRISPRa systems with unique properties, for example, ability to activate from specific positions upstream of a gene of interest. Here, we describe a protocol for designing, characterizing, and applying the modular CRISPRa system for gene activation in E. coli. We first focus on how to identify activating sites upstream of promoters and the cloning of the targeting sgRNA. We then describe how to perform a fluorescence experiment to evaluate activation of a single target site. Finally, we explain how to adapt the system to expand the target range and how to characterize the activation pattern obtained from different CRISPRa designs.
CRISPR-Cas 调控子为控制基因表达提供了一个极好的工具箱，这是因为其组件的多功能性和对靶向 DNA 序列的单指导 RNA (sgRNA) 的易于编程。CRISPR 激活 (CRISPRa) 系统也包含在其中。这些系统允许用户通过将转录激活结构域 (AD) 定位在启动子元件附近，从而激活靶基因的转录，从而招募 RNA 聚合酶 (RNAP) 启动转录。已经描述了多种不同的 CRISPRa 系统，它们在 AD 类型、募集策略和 CRISPR-Cas 系统方面有所不同。最近，描述了一种高度模块化的 CRISPRa 系统，该系统允许 AD 和 CRISPR-Cas 组件的方便交换。这允许创建具有独特特性的 CRISPRa 系统，例如，能够从感兴趣基因的上游特定位置激活的能力。在这里，我们描述了一种用于在大肠杆菌中设计、表征和应用模块化 CRISPRa 系统进行基因激活的方案。我们首先关注如何识别启动子上游的激活位点以及靶向 sgRNA 的克隆。然后，我们描述了如何进行荧光实验来评估单个靶位点的激活。最后，我们解释了如何适应系统以扩大目标范围，以及如何表征从不同 CRISPRa 设计获得的激活模式。
