Department of Immunology, Binzhou Medical University, Shandong, China.
State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China.
EMBO Rep. 2022 Apr 5;23(4):e53691. doi: 10.15252/embr.202153691. Epub 2022 Feb 24.
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.
揭示复杂生物过程中基因的功能对于系统生物学至关重要。然而，目前尚无简单可靠的实验工具可用于在单个实验中对每种可能组合的多个基因进行失活功能实验，这对于解析给定表型中多个基因的交互作用至关重要。在这项研究中，我们开发了 miR-AB，这是一种新型基于 microRNA 的 shRNA（shRNAmir）骨架，可简化、经济高效且无误地生产 shRNAmirs。在体外和体内验证其强大的 RNAi 效率后，miR-AB 被整合到一个包含多个真核启动子的病毒工具包中，以使其能够应用于多种细胞类型。我们进一步将八种发射整个可见光谱波长的荧光蛋白工程化到这个工具包中，并使用它来建立一个多色编码的多路复用 RNAi 测定法，其中多个基因可以在单细胞水平上单独和组合地被强烈和可靠地沉默。
