Construction and characterization of synthetic microRNA cluster for multiplex RNA interference in mammalian cells
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ABSTRACT: RNA interference (RNAi) technology is widely used in basic and translational research. By mimicking natural primary microRNA (pri-miRNA) cluster, multiple engineered hairpins can be transcribed as a single transcript from the same Pol-II promoter, enabling multiplex RNAi in mammalian cells 1-5. However, constructing synthetic miRNA cluster is still time-consuming, and the processing and function of miRNA cluster are incompletely understood. Here, we identified a miRNA precursor architecture that allowed precise miRNA maturation. We established a hierarchical cloning method for efficient construction of synthetic miRNA cluster harboring up to 18 miRNA precursors. We demonstrated that maturation and function of individual miRNA precursors were independent on their positions in the cluster. We then analyzed the integration efficiency of miRNA clusters with varying number of miRNA precursors by using CRISPR/Cas9 mediated integration, piggyBac transposon system, and lentiviral system. This synthetic miRNA cluster system provides an important tool for multiplex RNAi in mammalian cells.
ORGANISM(S): Homo sapiens
PROVIDER: GSE71088 | GEO | 2016/09/01
SECONDARY ACCESSION(S): PRJNA290333
REPOSITORIES: GEO
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