National and Local Joint Engineering Laboratory of Medical Synthetic Biology, Shenzhen, Second People's Hospital, The First Affiliated Hospital of Shenzhen University Health Science Center, Shenzhen518035, China.
Department of Urology, Shenzhen Second People's Hospital, The First Affiliated Hospital of Shenzhen University Health Science Center, Shenzhen518035, China.
Theranostics. 2020 May 17;10(15):6661-6673. doi: 10.7150/thno.44880. eCollection 2020.
CRISPR-Cas9 has become a versatile tool for genome editing and regulation, and strategies to effectively control its activity have attracted much attention. RNAi, also a gene-regulating tool, is used as another mechanism by which eukaryotes resist the invasion of foreign genetic material. : In this study, we analyzed the quantitative inhibition of the CRISPR system by using artificial miRNAs (amiRNAs) combined with the RNAi enhancer enoxacin to improve the targeting specificity of the CRISPR system. Furthermore, we examined the feasibility of improving the efficiency of gene editing and regulation by blocking the effects of natural intracellular miRNAs on sgRNAs. : amiRNAs targeting the sgRNA were used to control its expression, and the small molecule drug denoxacin was utilized to enhance this effect, especially in the presence of Cas9. amiRNA/enoxacin inhibited CRISPR-mediated gene editing and regulation both and and could tune sgRNA-targeting specificity. Furthermore, CRISPR efficiency was increased by blocking the effects of endogenous miRNAs. : Our study provides an efficient molecular switch for conditional regulation of CRISPR activities in mammalian cells and also presents potentially useful approaches for solving current key issues of off-target effects and low targeting efficiency.
CRISPR-Cas9 已成为基因组编辑和调控的通用工具，有效控制其活性的策略引起了广泛关注。RNAi 也是一种基因调控工具，被真核生物用来抵抗外来遗传物质的入侵。在这项研究中，我们分析了利用人工 microRNA（amiRNA）结合 RNAi 增强剂恩诺沙星抑制 CRISPR 系统的定量抑制作用，以提高 CRISPR 系统的靶向特异性。此外，我们还研究了通过阻断天然细胞内 microRNA 对 sgRNA 的影响来提高基因编辑和调控效率的可行性。针对 sgRNA 的 amiRNA 用于控制其表达，小分子药物恩诺沙星增强了这种作用，特别是在 Cas9 存在的情况下。amiRNA/恩诺沙星抑制了 CRISPR 介导的基因编辑和调控，既能调节 sgRNA 的靶向特异性。此外，通过阻断内源性 miRNAs 的作用可以提高 CRISPR 的效率。本研究为哺乳动物细胞中 CRISPR 活性的条件调节提供了一种有效的分子开关，也为解决当前关键的脱靶效应和低靶向效率问题提供了潜在的有用方法。
