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

Basic biological research and biomedical applications often require studying the multiple interactions between genes or proteins while multiplex RNA interference (RNAi) technology is still challenging in mammalian cells. In mammalian genomes, the natural microRNA (miRNA) clusters, of which the miRNAs often share similar expression patterns and target diverse genes, would provide a potential multiplex RNAi scaffold. Based on the natural pri-miR-155 precursor, we have developed and characterized a multiplex RNAi method by engineering synthetic miRNA clusters, among which the maturation and function of individual miRNA precursors are independent of their positions in the cluster. And the synthetic miRNA clusters are assembled by an efficient hierarchical Golden-Gate cloning method. Here, we describe the design rules and the hierarchical cloning methods to construct synthetic miRNA cluster, and the brief protocol for the integration of synthetic miRNA clusters into the mammalian genome.