Construction and Characterization of a Synthetic MicroRNA Cluster for Multiplex RNA Interference in Mammalian Cells

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