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Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy
Published on: October 4, 2019
Construction and Characterization of a Synthetic MicroRNA Cluster for Multiplex RNA Interference in Mammalian Cells
Tingting Wang1, Yue Xie1, Aidi Tan1
1MOE Key Laboratory of Bioinformatics and Bioinformatics Division, Center for Synthetic and Systems Biology, TNLIST/Department of Automation, Tsinghua University , Beijing 100084, China.
Abstract:
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.
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