miR‐AB, a miRNA‐based shRNA viral toolkit for multicolor‐barcoded multiplex RNAi at a single‐cell level

Uncovering the functions of genes in a complex biological process is fundamental for systems biology. However, currently there is no simple and reliable experimental tool available to conduct loss‐of‐function experiments for multiple genes in every possible combination in a single experiment, which is vital for parsing the interactive role of multiple genes in a given phenotype. In this study, we develop miR‐AB, a new microRNA‐based shRNA (shRNAmir) backbone for simplified, cost‐effective, and error‐proof production of shRNAmirs. After verification of its potent RNAi efficiency in vitro and in vivo, miR‐AB was integrated into a viral toolkit containing multiple eukaryotic promoters to enable its application in diverse cell types. We further engineer eight fluorescent proteins emitting wavelengths across the entire visible spectrum into this toolkit and use it to set up a multicolor‐barcoded multiplex RNAi assay where multiple genes are strongly and reliably silenced both individually and combinatorially at a single‐cell level.