FAMSi: A Synthetic Biology Approach to the Fast Assembly of Multiplex siRNAs for Silencing Gene Expression in Mammalian Cells
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,...
| Main Authors: | , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2020-12-01
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| Series: | Molecular Therapy: Nucleic Acids |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S216225312030319X |
| _version_ | 1831544599119659008 |
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| author | 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 |
| author_facet | 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 |
| author_sort | Fang He |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-17T01:12:54Z |
| format | Article |
| id | doaj.art-6d0374a049b84e5ab294d9468447091c |
| institution | Directory Open Access Journal |
| issn | 2162-2531 |
| language | English |
| last_indexed | 2024-12-17T01:12:54Z |
| publishDate | 2020-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj.art-6d0374a049b84e5ab294d9468447091c2022-12-21T22:09:06ZengElsevierMolecular Therapy: Nucleic Acids2162-25312020-12-0122885899FAMSi: A Synthetic Biology Approach to the Fast Assembly of Multiplex siRNAs for Silencing Gene Expression in Mammalian CellsFang He0Na Ni1Zongyue Zeng2Di Wu3Yixiao Feng4Alexander J. Li5Benjamin Luu6Alissa F. Li7Kevin Qin8Eric Wang9Xi Wang10Xiaoxing Wu11Huaxiu Luo12Jing Zhang13Meng Zhang14Yukun Mao15Mikhail Pakvasa16William Wagstaff17Yongtao Zhang18Changchun Niu19Hao Wang20Linjuan Huang21Deyao Shi22Qing Liu23Xia Zhao24Kai Fu25Russell R. Reid26Jennifer Moriatis Wolf27Michael J. Lee28Kelly Hynes29Jason Strelzow30Mostafa El Dafrawy31Hua Gan32Tong-Chuan He33Jiaming Fan34Ministry 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, ChinaMolecular 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, ChinaMinistry 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, USAMinistry 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, USAMolecular 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, ChinaMolecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USAMolecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USAMolecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USAMolecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USAMolecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USAMinistry 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, USAMolecular 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, ChinaMolecular 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, ChinaMolecular 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, ChinaMolecular 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, ChinaMolecular 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, ChinaMolecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USAMolecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USAMolecular 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, ChinaMolecular 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, ChinaMinistry 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, USAMolecular 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, ChinaMolecular 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, ChinaMolecular 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, ChinaMolecular 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, ChinaMolecular 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, ChinaMolecular 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, USAMolecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USAMolecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USAMolecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USAMolecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USAMolecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USAMinistry of Education Key Laboratory of Diagnostic Medicine, School of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, ChinaMolecular 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.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.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.http://www.sciencedirect.com/science/article/pii/S216225312030319XRNA interferenceRNAidouble-stranded small interfering RNAsiRNABMP9/Smad signalingmesenchymal stem cells |
| spellingShingle | 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 FAMSi: A Synthetic Biology Approach to the Fast Assembly of Multiplex siRNAs for Silencing Gene Expression in Mammalian Cells Molecular Therapy: Nucleic Acids RNA interference RNAi double-stranded small interfering RNA siRNA BMP9/Smad signaling mesenchymal stem cells |
| title | FAMSi: A Synthetic Biology Approach to the Fast Assembly of Multiplex siRNAs for Silencing Gene Expression in Mammalian Cells |
| title_full | FAMSi: A Synthetic Biology Approach to the Fast Assembly of Multiplex siRNAs for Silencing Gene Expression in Mammalian Cells |
| title_fullStr | FAMSi: A Synthetic Biology Approach to the Fast Assembly of Multiplex siRNAs for Silencing Gene Expression in Mammalian Cells |
| title_full_unstemmed | FAMSi: A Synthetic Biology Approach to the Fast Assembly of Multiplex siRNAs for Silencing Gene Expression in Mammalian Cells |
| title_short | FAMSi: A Synthetic Biology Approach to the Fast Assembly of Multiplex siRNAs for Silencing Gene Expression in Mammalian Cells |
| title_sort | famsi a synthetic biology approach to the fast assembly of multiplex sirnas for silencing gene expression in mammalian cells |
| topic | RNA interference RNAi double-stranded small interfering RNA siRNA BMP9/Smad signaling mesenchymal stem cells |
| url | http://www.sciencedirect.com/science/article/pii/S216225312030319X |
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