miR‐AB,a miRNA‐based shRNA viral toolkit for multicolor‐barcoded multiplex RNAi at a single‐cell level |
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Authors: | Dapeng Wang Jianbo Xiu Jiangyue Zhao Junli Luo |
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Affiliation: | 1. Department of Immunology, Binzhou Medical University, Shandong China ; 2. State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing China ; 3. Department of Ophthalmology, The 4th Affiliated Hospital of China Medical University, Shenyang China ; 4. Department of Molecular Medicine, The Scripps Research Institute, Jupiter FL, USA |
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Abstract: | 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. |
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Keywords: | multicolor multiplex RNAi shRNA shRNAmir |
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