Molecular Therapy: Nucleic Acids (Dec 2020)
FAMSi: A Synthetic Biology Approach to the Fast Assembly of Multiplex siRNAs for Silencing Gene Expression in Mammalian Cells
- Fang He,
- Na Ni,
- Zongyue Zeng,
- Di Wu,
- Yixiao Feng,
- Alexander J. Li,
- Benjamin Luu,
- Alissa F. Li,
- Kevin Qin,
- Eric Wang,
- Xi Wang,
- Xiaoxing Wu,
- Huaxiu Luo,
- Jing Zhang,
- Meng Zhang,
- Yukun Mao,
- Mikhail Pakvasa,
- William Wagstaff,
- Yongtao Zhang,
- Changchun Niu,
- Hao Wang,
- Linjuan Huang,
- Deyao Shi,
- Qing Liu,
- Xia Zhao,
- Kai Fu,
- Russell R. Reid,
- Jennifer Moriatis Wolf,
- Michael J. Lee,
- Kelly Hynes,
- Jason Strelzow,
- Mostafa El Dafrawy,
- Hua Gan,
- Tong-Chuan He,
- Jiaming Fan
Affiliations
- Fang He
- Ministry of Education Key Laboratory of Diagnostic Medicine, School of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China; Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Departments of Nephrology, Breast Surgery, Gastrointestinal Surgery, and Obstetrics and Gynecology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
- Na Ni
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Departments of Nephrology, Breast Surgery, Gastrointestinal Surgery, and Obstetrics and Gynecology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
- Zongyue Zeng
- Ministry of Education Key Laboratory of Diagnostic Medicine, School of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China; Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Di Wu
- Ministry of Education Key Laboratory of Diagnostic Medicine, School of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China; Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Yixiao Feng
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Departments of Nephrology, Breast Surgery, Gastrointestinal Surgery, and Obstetrics and Gynecology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
- Alexander J. Li
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Benjamin Luu
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Alissa F. Li
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Kevin Qin
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Eric Wang
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Xi Wang
- Ministry of Education Key Laboratory of Diagnostic Medicine, School of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China; Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Xiaoxing Wu
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Departments of Nephrology, Breast Surgery, Gastrointestinal Surgery, and Obstetrics and Gynecology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
- Huaxiu Luo
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Department of Burn and Plastic Surgery, West China Hospital of Sichuan University, Chengdu 610041, China
- Jing Zhang
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Departments of Nephrology, Breast Surgery, Gastrointestinal Surgery, and Obstetrics and Gynecology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
- Meng Zhang
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Department of Orthopaedic Surgery, The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510405, China
- Yukun Mao
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Departments of Orthopaedic Surgery and Neurosurgery, The Affiliated Zhongnan Hospital of Wuhan University, Wuhan 430072, China
- Mikhail Pakvasa
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- William Wagstaff
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Yongtao Zhang
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Department of Orthopaedic Surgery, The Affiliated Hospital of Qingdao University, Qingdao 266061, China
- Changchun Niu
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Department of Laboratory Diagnostic Medicine, The Affiliated Hospital of the University of Chinese Academy of Sciences, and Chongqing General Hospital, Chongqing 400021, China
- Hao Wang
- Ministry of Education Key Laboratory of Diagnostic Medicine, School of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China; Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Linjuan Huang
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Departments of Nephrology, Breast Surgery, Gastrointestinal Surgery, and Obstetrics and Gynecology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
- Deyao Shi
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Department of Orthopaedic Surgery, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
- Qing Liu
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Department of Spine Surgery, Second Xiangya Hospital, Central South University, Changsha 410011, China
- Xia Zhao
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Department of Orthopaedic Surgery, The Affiliated Hospital of Qingdao University, Qingdao 266061, China
- Kai Fu
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Departments of Orthopaedic Surgery and Neurosurgery, The Affiliated Zhongnan Hospital of Wuhan University, Wuhan 430072, China
- Russell R. Reid
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Department of Surgery Section of Plastic Surgery, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Jennifer Moriatis Wolf
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Michael J. Lee
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Kelly Hynes
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Jason Strelzow
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Mostafa El Dafrawy
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Hua Gan
- Ministry of Education Key Laboratory of Diagnostic Medicine, School of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China
- Tong-Chuan He
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Corresponding author: Tong-Chuan He, MD, PhD, Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, 5841 South Maryland Avenue, MC3079, Chicago, IL 60637, USA.
- Jiaming Fan
- Ministry of Education Key Laboratory of Diagnostic Medicine, School of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China; Corresponding author: Jiaming Fan, MD, PhD, Ministry of Education Key Laboratory of Diagnostic Medicine, School of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.
- Journal volume & issue
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Vol. 22
pp. 885 – 899
Abstract
RNA interference (RNAi) is mediated by an ∼21-nt double-stranded small interfering RNA (siRNA) and shows great promise in delineating gene functions and in developing therapeutics for human diseases. However, effective gene silencing usually requires the delivery of multiple siRNAs for a given gene, which is often technically challenging and time-consuming. In this study, by exploiting the type IIS restriction endonuclease-based synthetic biology methodology, we developed the fast assembly of multiplex siRNAs (FAMSi) system. In our proof-of-concept experiments, we demonstrated that multiple fragments containing three, four, or five siRNA sites targeting common Smad4 and/or BMPR-specific Smad1, Smad5, and Smad8 required for BMP9 signaling could be assembled efficiently. The constructed multiplex siRNAs effectively knocked down the expression of Smad4 and/or Smad1, Smad5, and Smad8 in mesenchymal stem cells (MSCs), and they inhibited all aspects of BMP9-induced osteogenic differentiation in bone marrow MSCs (BMSCs), including decreased expression of osteogenic regulators/markers, reduced osteogenic marker alkaline phosphatase (ALP) activity, and diminished in vitro matrix mineralization and in vivo ectopic bone formation. Collectively, we demonstrate that the engineered FAMSi system provides a fast-track platform for assembling multiplexed siRNAs in a single vector, and thus it may be a valuable tool to study gene functions or to develop novel siRNA-based therapeutics.
