Wang Tingting, Xie Yue, Tan Aidi, Li Shao, Xie Zhen
MOE Key Laboratory of Bioinformatics and Bioinformatics Division, Center for Synthetic and Systems Biology, TNLIST/Department of Automation, Tsinghua University , Beijing 100084, China.
ACS Synth Biol. 2016 Nov 18;5(11):1193-1200. doi: 10.1021/acssynbio.5b00180. Epub 2015 Dec 22.
RNA interference (RNAi) technology is widely used in basic and translational research. By mimicking a natural primary microRNA (pri-miRNA) cluster, multiple engineered hairpins can be transcribed as a single transcript from the same Pol II promoter, enabling the formation of multiplex RNAi in mammalian cells. However, constructing a synthetic miRNA cluster is still time-consuming, and the processing and function of a miRNA cluster are incompletely understood. Here, we identified a miRNA precursor architecture that allows precise miRNA maturation. We established a hierarchical cloning method for the efficient construction of a synthetic miRNA cluster harboring up to 18 miRNA precursors. We demonstrated that the maturation and function of individual miRNA precursors were independent of their positions in the cluster. We then analyzed the integration efficiency of miRNA clusters having a varied number of miRNA precursors by using CRISPR/Cas9-mediated integration, a piggyBac transposon system, and a lentiviral system. This synthetic miRNA cluster system provides an important tool for multiplex RNAi in mammalian cells.
RNA干扰（RNAi）技术广泛应用于基础研究和转化研究。通过模拟天然初级微小RNA（pri-miRNA）簇，多个工程化发夹可以从同一个Pol II启动子转录为单个转录本，从而在哺乳动物细胞中形成多重RNAi。然而，构建合成miRNA簇仍然耗时，并且miRNA簇的加工和功能尚未完全了解。在这里，我们鉴定了一种允许精确miRNA成熟的miRNA前体结构。我们建立了一种分层克隆方法，用于高效构建包含多达18个miRNA前体的合成miRNA簇。我们证明了单个miRNA前体的成熟和功能与其在簇中的位置无关。然后，我们通过使用CRISPR/Cas9介导的整合、piggyBac转座子系统和慢病毒系统，分析了具有不同数量miRNA前体的miRNA簇的整合效率。这种合成miRNA簇系统为哺乳动物细胞中的多重RNAi提供了一个重要工具。
