Developing a Versatile Shotgun Cloning Strategy for Single-Vector-Based Multiplex Expression of Short Interfering RNAs (siRNAs) in Mammalian Cells

• Published in: ACS synthetic biology Vol. 8; no. 9; pp. 2092 - 2105
• Main Authors: Wang, Xi, Yuan, Chengfu, Huang, Bo, Fan, Jiaming, Feng, Yixiao, Li, Alexander J, Zhang, Bo, Lei, Yan, Ye, Zhenyu, Zhao, Ling, Cao, Daigui, Yang, Lijuan, Wu, Di, Chen, Xian, Liu, Bin, Wagstaff, William, He, Fang, Wu, Xiaoxing, Luo, Huaxiu, Zhang, Jing, Zhang, Meng, Haydon, Rex C, Luu, Hue H, Lee, Michael J, Moriatis Wolf, Jennifer, Huang, Ailong, He, Tong-Chuan, Zeng, Zongyue
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 20.09.2019
• Subjects: Wnt/β-catenin signaling; RNA interference (RNAi); BMP9/Smad4 signaling; mesenchymal stem cells (MSCs); osteogenic differentiation; multiplex expression; short interfering double-stranded RNA (siRNA); shotgun cloning
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.9b00203

As an important post-transcriptional regulatory machinery mediated by ∼21nt short-interfering double-stranded RNA (siRNA), RNA interference (RNAi) is a powerful tool to delineate gene functions and develop therapeutics. However, effective RNAi-mediated silencing requires multiple siRNAs for given genes, a time-consuming process to accomplish. Here, we developed a user-friendly system for single-vector-based multiplex siRNA expression by exploiting the unique feature of restriction endonuclease BstXI. Specifically, we engineered a BstXI-based shotgun cloning (BSG) system, which consists of three entry vectors with siRNA expression units (SiEUs) flanked with distinct BstXI sites, and a retroviral destination vector for shotgun SiEU assembly. For proof-of-principle studies, we constructed multiplex siRNA vectors silencing β-catenin and/or Smad4 and assessed their functionalities in mesenchymal stem cells (MSCs). Pooled siRNA cassettes were effectively inserted into respective entry vectors in one-step, and shotgun seamless assembly of pooled BstXI-digested SiEU fragments into a retroviral destination vector followed. We found these multiplex siRNAs effectively silenced β-catenin and/or Smad4, and inhibited Wnt3A- or BMP9-specific reporters and downstream target expression in MSCs. Furthermore, multiplex silencing of β-catenin and/or Smad4 diminished Wnt3A and/or BMP9-induced osteogenic differentiation. Collectively, the BSG system is a user-friendly technology for single-vector-based multiplex siRNA expression to study gene functions and develop experimental therapeutics.