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Diaby M, Wu H, Gao B, Shi S, Wang B, Wang S, Wang Y, Wu Z, Chen C, Wang X, Song C. A Naturally Active Spy Transposon Discovered from the Insect Genome of Colletes gigas as a Promising Novel Gene Transfer Tool. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2024; 11:e2400969. [PMID: 38774947 PMCID: PMC11304231 DOI: 10.1002/advs.202400969] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/31/2024] [Revised: 04/09/2024] [Indexed: 08/09/2024]
Abstract
Novel active DNA transposons, such as Spy transposons from the PHIS superfamily, are identified through bioinformatics in this study. The native transposases cgSpy and cvSpy displayed transposition activities of approximately 85% and 35% compared to the hyperactive piggyBac transposase (hyPB). The cgSpy transposon showed unique characteristics, including a lack of overproduction inhibition and reduced efficiency for insertion sizes between 3.1 to 8.5 kb. Integration preferences of cgSpy are found in genes and regulatory regions, making it suitable for genetic manipulation. Evaluation in T-cell engineering demonstrated that cgSpy-mediated chimeric antigen receptor (CAR) modification is comparable to the PB system, indicating its potential utility in cell therapy. This study unveils the promising application of the active native transposase, Spy, from Colletes gigas, as a valuable tool for genetic engineering, particularly in T-cell manipulation.
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Affiliation(s)
- Mohamed Diaby
- College of Animal Science & TechnologyYangzhou UniversityYangzhouJiangsu225009China
| | - Han Wu
- School of Basic Medical SciencesShenzhen University Medical SchoolShenzhen UniversityShenzhenGuangdong518055China
| | - Bo Gao
- College of Animal Science & TechnologyYangzhou UniversityYangzhouJiangsu225009China
| | - Shasha Shi
- College of Animal Science & TechnologyYangzhou UniversityYangzhouJiangsu225009China
| | - Bingqing Wang
- College of Animal Science & TechnologyYangzhou UniversityYangzhouJiangsu225009China
| | - Saisai Wang
- College of Animal Science & TechnologyYangzhou UniversityYangzhouJiangsu225009China
| | - Yali Wang
- College of Animal Science & TechnologyYangzhou UniversityYangzhouJiangsu225009China
| | - Zherui Wu
- School of Basic Medical SciencesShenzhen University Medical SchoolShenzhen UniversityShenzhenGuangdong518055China
| | - Cai Chen
- College of Animal Science & TechnologyYangzhou UniversityYangzhouJiangsu225009China
| | - Xiaoyan Wang
- College of Animal Science & TechnologyYangzhou UniversityYangzhouJiangsu225009China
| | - Chengyi Song
- College of Animal Science & TechnologyYangzhou UniversityYangzhouJiangsu225009China
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2
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Liu P, Panda K, Edwards SA, Swanson R, Yi H, Pandesha P, Hung YH, Klaas G, Ye X, Collins MV, Renken KN, Gilbertson LA, Veena V, Hancock CN, Slotkin RK. Transposase-assisted target-site integration for efficient plant genome engineering. Nature 2024; 631:593-600. [PMID: 38926583 PMCID: PMC11254759 DOI: 10.1038/s41586-024-07613-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2023] [Accepted: 05/28/2024] [Indexed: 06/28/2024]
Abstract
The current technologies to place new DNA into specific locations in plant genomes are low frequency and error-prone, and this inefficiency hampers genome-editing approaches to develop improved crops1,2. Often considered to be genome 'parasites', transposable elements (TEs) evolved to insert their DNA seamlessly into genomes3-5. Eukaryotic TEs select their site of insertion based on preferences for chromatin contexts, which differ for each TE type6-9. Here we developed a genome engineering tool that controls the TE insertion site and cargo delivered, taking advantage of the natural ability of the TE to precisely excise and insert into the genome. Inspired by CRISPR-associated transposases that target transposition in a programmable manner in bacteria10-12, we fused the rice Pong transposase protein to the Cas9 or Cas12a programmable nucleases. We demonstrated sequence-specific targeted insertion (guided by the CRISPR gRNA) of enhancer elements, an open reading frame and a gene expression cassette into the genome of the model plant Arabidopsis. We then translated this system into soybean-a major global crop in need of targeted insertion technology. We have engineered a TE 'parasite' into a usable and accessible toolkit that enables the sequence-specific targeting of custom DNA into plant genomes.
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Affiliation(s)
- Peng Liu
- Donald Danforth Plant Science Center, St Louis, MO, USA
| | - Kaushik Panda
- Donald Danforth Plant Science Center, St Louis, MO, USA
| | - Seth A Edwards
- Donald Danforth Plant Science Center, St Louis, MO, USA
- Division of Biological Sciences, University of Missouri, Columbia, MO, USA
| | - Ryan Swanson
- Donald Danforth Plant Science Center, St Louis, MO, USA
- Division of Biological Sciences, University of Missouri, Columbia, MO, USA
| | - Hochul Yi
- Plant Transformation Facility, Donald Danforth Plant Science Center, St Louis, MO, USA
| | - Pratheek Pandesha
- Donald Danforth Plant Science Center, St Louis, MO, USA
- Division of Biology and Biomedical Sciences, Washington University, St Louis, MO, USA
| | - Yu-Hung Hung
- Donald Danforth Plant Science Center, St Louis, MO, USA
| | - Gerald Klaas
- Donald Danforth Plant Science Center, St Louis, MO, USA
| | - Xudong Ye
- Bayer Crop Science, St Louis, MO, USA
| | | | | | | | - Veena Veena
- Plant Transformation Facility, Donald Danforth Plant Science Center, St Louis, MO, USA
| | | | - R Keith Slotkin
- Donald Danforth Plant Science Center, St Louis, MO, USA.
- Division of Biological Sciences, University of Missouri, Columbia, MO, USA.
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3
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Pacesa M, Pelea O, Jinek M. Past, present, and future of CRISPR genome editing technologies. Cell 2024; 187:1076-1100. [PMID: 38428389 DOI: 10.1016/j.cell.2024.01.042] [Citation(s) in RCA: 81] [Impact Index Per Article: 81.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2023] [Revised: 01/23/2024] [Accepted: 01/26/2024] [Indexed: 03/03/2024]
Abstract
Genome editing has been a transformative force in the life sciences and human medicine, offering unprecedented opportunities to dissect complex biological processes and treat the underlying causes of many genetic diseases. CRISPR-based technologies, with their remarkable efficiency and easy programmability, stand at the forefront of this revolution. In this Review, we discuss the current state of CRISPR gene editing technologies in both research and therapy, highlighting limitations that constrain them and the technological innovations that have been developed in recent years to address them. Additionally, we examine and summarize the current landscape of gene editing applications in the context of human health and therapeutics. Finally, we outline potential future developments that could shape gene editing technologies and their applications in the coming years.
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Affiliation(s)
- Martin Pacesa
- Laboratory of Protein Design and Immunoengineering, École Polytechnique Fédérale de Lausanne and Swiss Institute of Bioinformatics, Station 19, CH-1015 Lausanne, Switzerland
| | - Oana Pelea
- Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
| | - Martin Jinek
- Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
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4
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Lampe GD, King RT, Halpin-Healy TS, Klompe SE, Hogan MI, Vo PLH, Tang S, Chavez A, Sternberg SH. Targeted DNA integration in human cells without double-strand breaks using CRISPR-associated transposases. Nat Biotechnol 2024; 42:87-98. [PMID: 36991112 PMCID: PMC10620015 DOI: 10.1038/s41587-023-01748-1] [Citation(s) in RCA: 60] [Impact Index Per Article: 60.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2021] [Accepted: 03/13/2023] [Indexed: 03/31/2023]
Abstract
Conventional genome engineering with CRISPR-Cas9 creates double-strand breaks (DSBs) that lead to undesirable byproducts and reduce product purity. Here we report an approach for programmable integration of large DNA sequences in human cells that avoids the generation of DSBs by using Type I-F CRISPR-associated transposases (CASTs). We optimized DNA targeting by the QCascade complex through protein design and developed potent transcriptional activators by exploiting the multi-valent recruitment of the AAA+ ATPase TnsC to genomic sites targeted by QCascade. After initial detection of plasmid-based integration, we screened 15 additional CAST systems from a wide range of bacterial hosts, identified a homolog from Pseudoalteromonas that exhibits improved activity and further increased integration efficiencies. Finally, we discovered that bacterial ClpX enhances genomic integration by multiple orders of magnitude, likely by promoting active disassembly of the post-integration CAST complex, akin to its known role in Mu transposition. Our work highlights the ability to reconstitute complex, multi-component machineries in human cells and establishes a strong foundation to exploit CRISPR-associated transposases for eukaryotic genome engineering.
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Affiliation(s)
- George D Lampe
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA
| | - Rebeca T King
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA
| | - Tyler S Halpin-Healy
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA
- Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA
| | - Sanne E Klompe
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA
- Department of Genomes and Genetics, Institut Pasteur, Paris, France
| | - Marcus I Hogan
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA
- Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA, USA
| | - Phuc Leo H Vo
- Department of Molecular Pharmacology and Therapeutics, Columbia University, New York, NY, USA
- Vertex Pharmaceuticals, Inc., Boston, MA, USA
| | - Stephen Tang
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA
| | - Alejandro Chavez
- Department of Pathology and Cell Biology, Columbia University, New York, NY, USA
- Department of Pediatrics, University of California, San Diego, La Jolla, CA, USA
| | - Samuel H Sternberg
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.
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5
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Tou CJ, Kleinstiver BP. Recent Advances in Double-Strand Break-Free Kilobase-Scale Genome Editing Technologies. Biochemistry 2023; 62:3493-3499. [PMID: 36049184 PMCID: PMC10239562 DOI: 10.1021/acs.biochem.2c00311] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
Genome editing approaches have transformed the ability to make user-defined changes to genomes in both ex vivo and in vivo contexts. Despite the abundant development of technologies that permit the installation of nucleotide-level changes, until recently, larger-scale sequence edits via technologies independent of DNA double-strand breaks (DSBs) had remained less explored. Here, we review recent advances toward DSB-free technologies that enable kilobase-scale modifications including insertions, deletions, inversions, replacements, and others. These technologies provide new capabilities for users, while offering hope for the simplification of putative therapeutic strategies by moving away from small mutation-specific edits and toward more generalizable kilobase-scale approaches.
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Affiliation(s)
- Connor J. Tou
- Biological Engineering Program, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA
- Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
- Department of Pathology, Massachusetts General Hospital, Boston, MA, 02114, USA
| | - Benjamin P. Kleinstiver
- Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA
- Department of Pathology, Massachusetts General Hospital, Boston, MA, 02114, USA
- Department of Pathology, Harvard Medical School, Boston, MA, 02115, USA
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6
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Han M, Perkins MH, Novaes LS, Xu T, Chang H. Advances in transposable elements: from mechanisms to applications in mammalian genomics. Front Genet 2023; 14:1290146. [PMID: 38098473 PMCID: PMC10719622 DOI: 10.3389/fgene.2023.1290146] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2023] [Accepted: 11/13/2023] [Indexed: 12/17/2023] Open
Abstract
It has been 70 years since Barbara McClintock discovered transposable elements (TE), and the mechanistic studies and functional applications of transposable elements have been at the forefront of life science research. As an essential part of the genome, TEs have been discovered in most species of prokaryotes and eukaryotes, and the relative proportion of the total genetic sequence they comprise gradually increases with the expansion of the genome. In humans, TEs account for about 40% of the genome and are deeply involved in gene regulation, chromosome structure maintenance, inflammatory response, and the etiology of genetic and non-genetic diseases. In-depth functional studies of TEs in mammalian cells and the human body have led to a greater understanding of these fundamental biological processes. At the same time, as a potent mutagen and efficient genome editing tool, TEs have been transformed into biological tools critical for developing new techniques. By controlling the random insertion of TEs into the genome to change the phenotype in cells and model organisms, critical proteins of many diseases have been systematically identified. Exploiting the TE's highly efficient in vitro insertion activity has driven the development of cutting-edge sequencing technologies. Recently, a new technology combining CRISPR with TEs was reported, which provides a novel targeted insertion system to both academia and industry. We suggest that interrogating biological processes that generally depend on the actions of TEs with TEs-derived genetic tools is a very efficient strategy. For example, excessive activation of TEs is an essential factor in the occurrence of cancer in humans. As potent mutagens, TEs have also been used to unravel the key regulatory elements and mechanisms of carcinogenesis. Through this review, we aim to effectively combine the traditional views of TEs with recent research progress, systematically link the mechanistic discoveries of TEs with the technological developments of TE-based tools, and provide a comprehensive approach and understanding for researchers in different fields.
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Affiliation(s)
- Mei Han
- Guangzhou National Laboratory, Guangzhou, China
| | - Matthew H. Perkins
- Nash Family Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY, United States
| | - Leonardo Santana Novaes
- Nash Family Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY, United States
| | - Tao Xu
- Guangzhou National Laboratory, Guangzhou, China
| | - Hao Chang
- Nash Family Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY, United States
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7
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Kovač A, Miskey C, Ivics Z. Sleeping Beauty Transposon Insertions into Nucleolar DNA by an Engineered Transposase Localized in the Nucleolus. Int J Mol Sci 2023; 24:14978. [PMID: 37834425 PMCID: PMC10573994 DOI: 10.3390/ijms241914978] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2023] [Revised: 09/22/2023] [Accepted: 09/28/2023] [Indexed: 10/15/2023] Open
Abstract
Transposons are nature's gene delivery vehicles that can be harnessed for experimental and therapeutic purposes. The Sleeping Beauty (SB) transposon shows efficient transposition and long-term transgene expression in human cells, and is currently under clinical development for gene therapy. SB transposition occurs into the human genome in a random manner, which carries a risk of potential genotoxic effects associated with transposon integration. Here, we evaluated an experimental strategy to manipulate SB's target site distribution by preferentially compartmentalizing the SB transposase to the nucleolus, which contains repetitive ribosomal RNA (rRNA) genes. We generated a fusion protein composed of the nucleolar protein nucleophosmin (B23) and the SB100X transposase, which was found to retain almost full transposition activity as compared to unfused transposase and to be predominantly localized to nucleoli in transfected human cells. Analysis of transposon integration sites generated by B23-SB100X revealed a significant enrichment into the p-arms of chromosomes containing nucleolus organizing regions (NORs), with preferential integration into the p13 and p11.2 cytobands directly neighboring the NORs. This bias in the integration pattern was accompanied by an enrichment of insertions into nucleolus-associated chromatin domains (NADs) at the periphery of nucleolar DNA and into lamina-associated domains (LADs). Finally, sub-nuclear targeting of the transposase resulted in preferential integration into chromosomal domains associated with the Upstream Binding Transcription Factor (UBTF) that plays a critical role in the transcription of 47S rDNA gene repeats of the NORs by RNA Pol I. Future modifications of this technology may allow the development of methods for specific gene insertion for precision genetic engineering.
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Affiliation(s)
| | | | - Zoltán Ivics
- Transposition and Genome Engineering, Research Centre of the Division of Hematology, Gene and Cell Therapy, Paul Ehrlich Institute, Paul Ehrlich Str. 51–59, D-63225 Langen, Germany; (A.K.); (C.M.)
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8
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Yarnall MTN, Ioannidi EI, Schmitt-Ulms C, Krajeski RN, Lim J, Villiger L, Zhou W, Jiang K, Garushyants SK, Roberts N, Zhang L, Vakulskas CA, Walker JA, Kadina AP, Zepeda AE, Holden K, Ma H, Xie J, Gao G, Foquet L, Bial G, Donnelly SK, Miyata Y, Radiloff DR, Henderson JM, Ujita A, Abudayyeh OO, Gootenberg JS. Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases. Nat Biotechnol 2023; 41:500-512. [PMID: 36424489 PMCID: PMC10257351 DOI: 10.1038/s41587-022-01527-4] [Citation(s) in RCA: 209] [Impact Index Per Article: 104.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2022] [Accepted: 09/23/2022] [Indexed: 11/26/2022]
Abstract
Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We present programmable addition via site-specific targeting elements (PASTE), which uses a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We demonstrate integration of sequences as large as ~36 kilobases at multiple genomic loci across three human cell lines, primary T cells and non-dividing primary human hepatocytes. To augment PASTE, we discovered 25,614 serine integrases and cognate attachment sites from metagenomes and engineered orthologs with higher activity and shorter recognition sequences for efficient programmable integration. PASTE has editing efficiencies similar to or exceeding those of homology-directed repair and non-homologous end joining-based methods, with activity in non-dividing cells and in vivo with fewer detectable off-target events. PASTE expands the capabilities of genome editing by allowing large, multiplexed gene insertion without reliance on DNA repair pathways.
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Affiliation(s)
- Matthew T N Yarnall
- McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Eleonora I Ioannidi
- McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA
- ETH Zürich, Zürich, Switzerland
| | - Cian Schmitt-Ulms
- McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Rohan N Krajeski
- McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Justin Lim
- McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Lukas Villiger
- McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Wenyuan Zhou
- McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Kaiyi Jiang
- McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Sofya K Garushyants
- National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA
| | | | - Liyang Zhang
- Integrated DNA Technologies, Coralville, IA, USA
| | | | | | | | | | | | - Hong Ma
- University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Jun Xie
- University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Guangping Gao
- University of Massachusetts Chan Medical School, Worcester, MA, USA
| | | | - Greg Bial
- Yecuris Corporation, Tualatin, OR, USA
| | | | | | | | | | | | - Omar O Abudayyeh
- McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA.
| | - Jonathan S Gootenberg
- McGovern Institute for Brain Research at MIT, Massachusetts Institute of Technology, Cambridge, MA, USA.
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9
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Lampe GD, King RT, Halpin-Healy TS, Klompe SE, Hogan MI, Vo PLH, Tang S, Chavez A, Sternberg SH. Targeted DNA integration in human cells without double-strand breaks using CRISPR RNA-guided transposases. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.03.17.533036. [PMID: 36993517 PMCID: PMC10055298 DOI: 10.1101/2023.03.17.533036] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 06/19/2023]
Abstract
Traditional genome-editing reagents such as CRISPR-Cas9 achieve targeted DNA modification by introducing double-strand breaks (DSBs), thereby stimulating localized DNA repair by endogenous cellular repair factors. While highly effective at generating heterogenous knockout mutations, this approach suffers from undesirable byproducts and an inability to control product purity. Here we develop a system in human cells for programmable, DSB-free DNA integration using Type I CRISPR-associated transposons (CASTs). To adapt our previously described CAST systems, we optimized DNA targeting by the QCascade complex through a comprehensive assessment of protein design, and we developed potent transcriptional activators by exploiting the multi-valent recruitment of the AAA+ ATPase, TnsC, to genomic sites targeted by QCascade. After initial detection of plasmid-based transposition, we screened 15 homologous CAST systems from a wide range of bacterial hosts, identified a CAST homolog from Pseudoalteromonas that exhibited improved activity, and increased integration efficiencies through parameter optimization. We further discovered that bacterial ClpX enhances genomic integration by multiple orders of magnitude, and we propose that this critical accessory factor functions to drive active disassembly of the post-transposition CAST complex, akin to its demonstrated role in Mu transposition. Our work highlights the ability to functionally reconstitute complex, multi-component machineries in human cells, and establishes a strong foundation to realize the full potential of CRISPR-associated transposons for human genome engineering.
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Affiliation(s)
- George D Lampe
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA
| | - Rebeca T King
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA
| | - Tyler S Halpin-Healy
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA
| | - Sanne E Klompe
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA
| | - Marcus I Hogan
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA
| | - Phuc Leo H Vo
- Department of Molecular Pharmacology and Therapeutics, Columbia University, New York, NY, USA
| | - Stephen Tang
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA
| | - Alejandro Chavez
- Department of Pathology and Cell Biology, Columbia University, New York, NY, USA
| | - Samuel H Sternberg
- Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA
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10
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de Rossi J, Arefeayne Y, Robinson A, Segatori L. Emerging technologies for genetic control systems in cellular therapies. Curr Opin Biotechnol 2022; 78:102833. [PMID: 36334445 PMCID: PMC10091305 DOI: 10.1016/j.copbio.2022.102833] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2022] [Revised: 09/23/2022] [Accepted: 10/02/2022] [Indexed: 11/06/2022]
Abstract
Progress in synthetic biology has enabled the construction of designer cells that sense biological inputs, and, in response, activate user-defined biomolecular programs. Such engineered cells provide unique opportunities for treating a wide variety of diseases. Current strategies mostly rely on cell-surface receptor systems engineered to convert binding interactions into activation of a transcriptional program. Genetic control systems are emerging as an appealing alternative to receptor-based sensors as they overcome the need for receptor engineering and result in cellular behaviors that operate over therapeutically relevant timescales. Genetic control systems include synthetic gene networks, RNA-based sensors, and post-translational tools. These technologies present fundamental challenges, including the requirement for precise integration with innate pathways, the need for parts orthogonal to existing circuitries, and the metabolic burden induced by such complex cell engineering endeavors. This review discusses the challenges in the design of genetic control systems for cellular therapies and their translational applications.
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Affiliation(s)
- Jacopo de Rossi
- Systems, Synthetic, and Physical Biology, Rice University, Houston, United States
| | - Yafet Arefeayne
- Department of Bioengineering, Rice University, Houston, United States
| | - Ashley Robinson
- Systems, Synthetic, and Physical Biology, Rice University, Houston, United States
| | - Laura Segatori
- Systems, Synthetic, and Physical Biology, Rice University, Houston, United States; Department of Bioengineering, Rice University, Houston, United States; Department of Chemical and Biomolecular Engineering, Rice University, Houston, United States; Department of Biosciences, Rice University, Houston, United States.
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11
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Transposase-CRISPR mediated targeted integration (TransCRISTI) in the human genome. Sci Rep 2022; 12:3390. [PMID: 35232993 PMCID: PMC8888626 DOI: 10.1038/s41598-022-07158-8] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2021] [Accepted: 02/08/2022] [Indexed: 11/08/2022] Open
Abstract
Various methods have been used in targeted gene knock-in applications. CRISPR-based knock-in strategies based on homology-independent repair pathways such as CRISPR HITI have been shown to possess the best efficiency for gene knock-in in mammalian cells. However, these methods suffer from the probability of plasmid backbone insertion at the target site. On the other hand, studies trying to combine the targeting ability of the Cas9 molecule and the excision/integration capacity of the PB transposase have shown random integrations. In this study, we introduce a new homology-independent knock-in strategy, Transposase-CRISPR mediated Targeted Integration (TransCRISTI), that exploits a fusion of Cas9 nuclease and a double mutant piggyBac transposase. In isogenic mammalian cell lines, we show that the TransCRISTI method demonstrates higher efficiency (72%) for site-specific insertions than the CRISPR HITI (44%) strategy. Application of the TransCRISTI method resulted in site-directed integration in 4.13% and 3.69% of the initially transfected population in the human AAVS1and PML loci, respectively, while the CRISPR HITI strategy resulted in site-directed integration in the PML locus in only 0.6% of cells. We also observed lower off-target and random insertions in the TransCRISTI group than the CRISPR HITI group. The TransCRISTI technology represents a great potential for the accurate and high-efficiency knock-in of the desired transposable elements into the predetermined genomic locations.
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12
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Find and cut-and-transfer (FiCAT) mammalian genome engineering. Nat Commun 2021; 12:7071. [PMID: 34862378 PMCID: PMC8642419 DOI: 10.1038/s41467-021-27183-x] [Citation(s) in RCA: 27] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2020] [Accepted: 11/09/2021] [Indexed: 12/26/2022] Open
Abstract
While multiple technologies for small allele genome editing exist, robust technologies for targeted integration of large DNA fragments in mammalian genomes are still missing. Here we develop a gene delivery tool (FiCAT) combining the precision of a CRISPR-Cas9 (find module), and the payload transfer efficiency of an engineered piggyBac transposase (cut-and-transfer module). FiCAT combines the functionality of Cas9 DNA scanning and targeting DNA, with piggyBac donor DNA processing and transfer capacity. PiggyBac functional domains are engineered providing increased on-target integration while reducing off-target events. We demonstrate efficient delivery and programmable insertion of small and large payloads in cellulo (human (Hek293T, K-562) and mouse (C2C12)) and in vivo in mouse liver. Finally, we evolve more efficient versions of FiCAT by generating a targeted diversity of 394,000 variants and undergoing 4 rounds of evolution. In this work, we develop a precise and efficient targeted insertion of multi kilobase DNA fragments in mammalian genomes. Mammalian genome engineering has advanced tremendously over the last decade, however there is still a need for robust gene writing with size scaling capacity. Here the authors present Find Cut-and-Transfer (FiCAT) technology to delivery large targeted payload insertion in cell lines and in vivo in mouse models.
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13
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The large bat Helitron DNA transposase forms a compact monomeric assembly that buries and protects its covalently bound 5'-transposon end. Mol Cell 2021; 81:4271-4286.e4. [PMID: 34403695 DOI: 10.1016/j.molcel.2021.07.028] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2021] [Revised: 07/23/2021] [Accepted: 07/23/2021] [Indexed: 12/22/2022]
Abstract
Helitrons are widespread eukaryotic DNA transposons that have significantly contributed to genome variability and evolution, in part because of their distinctive, replicative rolling-circle mechanism, which often mobilizes adjacent genes. Although most eukaryotic transposases form oligomers and use RNase H-like domains to break and rejoin double-stranded DNA (dsDNA), Helitron transposases contain a single-stranded DNA (ssDNA)-specific HUH endonuclease domain. Here, we report the cryo-electron microscopy structure of a Helitron transposase bound to the 5'-transposon end, providing insight into its multidomain architecture and function. The monomeric transposase forms a tightly packed assembly that buries the covalently attached cleaved end, protecting it until the second end becomes available. The structure reveals unexpected architectural similarity to TraI, a bacterial relaxase that also catalyzes ssDNA movement. The HUH active site suggests how two juxtaposed tyrosines, a feature of many replication initiators that use HUH nucleases, couple the conformational shift of an α-helix to control strand cleavage and ligation reactions.
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14
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Applications of piggyBac Transposons for Genome Manipulation in Stem Cells. Stem Cells Int 2021; 2021:3829286. [PMID: 34567130 PMCID: PMC8460389 DOI: 10.1155/2021/3829286] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2021] [Accepted: 08/16/2021] [Indexed: 12/20/2022] Open
Abstract
Transposons are mobile genetic elements in the genome. The piggyBac (PB) transposon system is increasingly being used for stem cell research due to its high transposition efficiency and seamless excision capacity. Over the past few decades, forward genetic screens based on PB transposons have been successfully established to identify genes associated with drug resistance and stem cell-related characteristics. Moreover, PB transposon is regarded as a promising gene therapy vector and has been used in some clinically relevant stem cells. Here, we review the recent progress on the basic biology of PB, highlight its applications in current stem cell research, and discuss its advantages and challenges.
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15
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Ichikawa M, Akamine H, Murata M, Ito S, Takayama K, Mizuguchi H. Generation of tetracycline-controllable CYP3A4-expressing Caco-2 cells by the piggyBac transposon system. Sci Rep 2021; 11:11670. [PMID: 34083621 PMCID: PMC8175591 DOI: 10.1038/s41598-021-91160-z] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2021] [Accepted: 05/24/2021] [Indexed: 01/03/2023] Open
Abstract
Caco-2 cells are widely used as an in vitro intestinal epithelial cell model because they can form a monolayer and predict drug absorption with high accuracy. However, Caco-2 cells hardly express cytochrome P450 (CYP), a drug-metabolizing enzyme. It is known that CYP3A4 is the dominant drug-metabolizing enzyme in human small intestine. In this study, we generated CYP3A4-expressing Caco-2 (CYP3A4-Caco-2) cells and attempted to establish a model that can simultaneously evaluate drug absorption and metabolism. CYP3A4-Caco-2 cells were generated by piggyBac transposon vectors. A tetracycline-controllable CYP3A4 expression cassette (tet-on system) was stably transduced into Caco-2 cells, thus regulating the levels of CYP3A4 expression depending on the doxycycline concentration. The CYP3A4 expression levels in CYP3A4-Caco-2 cells cultured in the presence of doxycycline were similar to or higher than those of adult small intestine. The CYP3A4-Caco-2 cells had enough ability to metabolize midazolam, a substrate of CYP3A4. CYP3A4 overexpression had no negative effects on cell proliferation, barrier function, and P-glycoprotein activity in Caco-2 cells. Thus, we succeeded in establishing Caco-2 cells with CYP3A4 metabolizing activity comparable to in vivo human intestinal tissue. This cell line would be useful in pharmaceutical studies as a model that can simultaneously evaluate drug absorption and metabolism.
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Affiliation(s)
- Moe Ichikawa
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka, 565-0871, Japan
| | - Hiroki Akamine
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka, 565-0871, Japan
| | - Michika Murata
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka, 565-0871, Japan
| | - Sumito Ito
- GenoMembrane Co., Ltd., 2-3-18 Namamugi, Tsurumi-ku, Yokohama, Kanagawa, 230-0052, Japan
| | - Kazuo Takayama
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka, 565-0871, Japan
| | - Hiroyuki Mizuguchi
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka, 565-0871, Japan.
- Laboratory of Hepatocyte Regulation, National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka, 567-0085, Japan.
- Global Center for Medical Engineering and Informatics, Osaka University, Suita, Osaka, 565-0871, Japan.
- Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives (OTRI), Osaka University, Suita, Osaka, 565-0871, Japan.
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16
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Sandoval-Villegas N, Nurieva W, Amberger M, Ivics Z. Contemporary Transposon Tools: A Review and Guide through Mechanisms and Applications of Sleeping Beauty, piggyBac and Tol2 for Genome Engineering. Int J Mol Sci 2021; 22:ijms22105084. [PMID: 34064900 PMCID: PMC8151067 DOI: 10.3390/ijms22105084] [Citation(s) in RCA: 72] [Impact Index Per Article: 18.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2021] [Revised: 04/30/2021] [Accepted: 05/05/2021] [Indexed: 01/19/2023] Open
Abstract
Transposons are mobile genetic elements evolved to execute highly efficient integration of their genes into the genomes of their host cells. These natural DNA transfer vehicles have been harnessed as experimental tools for stably introducing a wide variety of foreign DNA sequences, including selectable marker genes, reporters, shRNA expression cassettes, mutagenic gene trap cassettes, and therapeutic gene constructs into the genomes of target cells in a regulated and highly efficient manner. Given that transposon components are typically supplied as naked nucleic acids (DNA and RNA) or recombinant protein, their use is simple, safe, and economically competitive. Thus, transposons enable several avenues for genome manipulations in vertebrates, including transgenesis for the generation of transgenic cells in tissue culture comprising the generation of pluripotent stem cells, the production of germline-transgenic animals for basic and applied research, forward genetic screens for functional gene annotation in model species and therapy of genetic disorders in humans. This review describes the molecular mechanisms involved in transposition reactions of the three most widely used transposon systems currently available (Sleeping Beauty, piggyBac, and Tol2), and discusses the various parameters and considerations pertinent to their experimental use, highlighting the state-of-the-art in transposon technology in diverse genetic applications.
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Affiliation(s)
| | | | | | - Zoltán Ivics
- Correspondence: ; Tel.: +49-6103-77-6000; Fax: +49-6103-77-1280
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17
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Tong B, Dong H, Cui Y, Jiang P, Jin Z, Zhang D. The Versatile Type V CRISPR Effectors and Their Application Prospects. Front Cell Dev Biol 2021; 8:622103. [PMID: 33614630 PMCID: PMC7889808 DOI: 10.3389/fcell.2020.622103] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2020] [Accepted: 12/21/2020] [Indexed: 12/12/2022] Open
Abstract
The class II clustered regularly interspaced short palindromic repeats (CRISPR)–Cas systems, characterized by a single effector protein, can be further subdivided into types II, V, and VI. The application of the type II CRISPR effector protein Cas9 as a sequence-specific nuclease in gene editing has revolutionized this field. Similarly, Cas13 as the effector protein of type VI provides a convenient tool for RNA manipulation. Additionally, the type V CRISPR–Cas system is another valuable resource with many subtypes and diverse functions. In this review, we summarize all the subtypes of the type V family that have been identified so far. According to the functions currently displayed by the type V family, we attempt to introduce the functional principle, current application status, and development prospects in biotechnology for all major members.
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Affiliation(s)
- Baisong Tong
- School of Biological Engineering, Dalian Polytechnic University, Dalian, China.,Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.,Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, China
| | - Huina Dong
- Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.,Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, China
| | - Yali Cui
- Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.,Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, China
| | - Pingtao Jiang
- School of Biological Engineering, Dalian Polytechnic University, Dalian, China.,Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.,Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, China
| | - Zhaoxia Jin
- School of Biological Engineering, Dalian Polytechnic University, Dalian, China
| | - Dawei Zhang
- Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.,Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, China.,University of Chinese Academy of Sciences, Beijing, China
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18
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Goshayeshi L, Yousefi Taemeh S, Dehdilani N, Nasiri M, Ghahramani Seno MM, Dehghani H. CRISPR/dCas9-mediated transposition with specificity and efficiency of site-directed genomic insertions. FASEB J 2021; 35:e21359. [PMID: 33496003 DOI: 10.1096/fj.202001830rr] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2020] [Revised: 12/24/2020] [Accepted: 12/28/2020] [Indexed: 12/28/2022]
Abstract
The ability and efficiency of targeted nucleases to perform sequence replacements or insertions into the genome are limited. This limited efficiency for sequence replacements or insertions can be explained by the dependency on DNA repair pathways, the possibility of cellular toxicity, and unwanted activation of proto-oncogenes. The piggyBac (PB) transposase uses a very efficient enzymatic mechanism to integrate DNA fragments into the genome in a random manner. In this study, we fused an RNA-guided catalytically inactive Cas9 (dCas9) to the PB transposase and used dual sgRNAs to localize this molecule to specific genomic targets. We designed and used a promoter/reporter complementation assay to register and recover cells harboring-specific integrations, where only by complementation upon correct genomic integration, the reporter can be activated. Using an RNA-guided piggyBac transposase and dual sgRNAs, we were able to achieve site-directed integrations in the human ROSA26 safe harbor region in 0.32% of cells. These findings show that the methodology used in this study can be used for targeting genomic regions. An application for this finding could be in cancer cells to insert sequences into specific target regions that are intended to be destroyed, or to place promoter cargos behind the tumor suppressor genes to activate them.
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Affiliation(s)
- Lena Goshayeshi
- Division of Biotechnology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran
- Stem Cell Biology and Regenerative Medicine Research Group, Research Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran
| | - Sara Yousefi Taemeh
- Division of Biotechnology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran
- Stem Cell Biology and Regenerative Medicine Research Group, Research Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran
| | - Nima Dehdilani
- Division of Biotechnology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran
- Stem Cell Biology and Regenerative Medicine Research Group, Research Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran
| | - Mohammadreza Nasiri
- Recombinant Proteins Research Group, Research Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran
- Department of Animal Science, Ferdowsi University of Mashhad, Mashhad, Iran
| | - Mohammad M Ghahramani Seno
- Division of Biotechnology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran
- Department of Basic Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran
| | - Hesam Dehghani
- Division of Biotechnology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran
- Stem Cell Biology and Regenerative Medicine Research Group, Research Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran
- Department of Basic Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran
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19
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Ma S, Wang X, Hu Y, Lv J, Liu C, Liao K, Guo X, Wang D, Lin Y, Rong Z. Enhancing site-specific DNA integration by a Cas9 nuclease fused with a DNA donor-binding domain. Nucleic Acids Res 2020; 48:10590-10601. [PMID: 32986839 PMCID: PMC7544211 DOI: 10.1093/nar/gkaa779] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2020] [Revised: 09/04/2020] [Accepted: 09/09/2020] [Indexed: 12/20/2022] Open
Abstract
The CRISPR/Cas system is widely used for genome editing. However, robust and targeted insertion of a DNA segment remains a challenge. Here, we present a fusion nuclease (Cas9-N57) to enhance site-specific DNA integration via a fused DNA binding domain of Sleeping Beauty transposase to tether the DNA segment to the Cas9/sgRNA complex. The insertion was unidirectional and specific, and DNA fragments up to 12 kb in length were successfully integrated. As a test of the system, Cas9-N57 mediated the insertion of a CD19-specific chimeric antigen receptor (CD19-CAR) cassette into the AAVS1 locus in human T cells, and induced intrahepatic cholangiocarcinoma in mice by simultaneously mediating the insertion of oncogenic KrasG12D into the Rosa26 locus and disrupting Trp53 and Pten. Moreover, the nuclease-N57 fusion proteins based on AsCpf1 (AsCas12a) and CjCas9 exhibited similar activity. These findings demonstrate that CRISPR-associated nuclease-N57 protein fusion is a powerful tool for targeted DNA insertion and holds great potential for gene therapy applications.
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Affiliation(s)
- Shufeng Ma
- Department of Nephrology, Shenzhen Hospital, Southern Medical University, Shenzhen 518110, China
- Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Xinlong Wang
- Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Yongfei Hu
- Dermatology Hospital, Southern Medical University, Guangzhou 510091, China
| | - Jie Lv
- Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Chengfang Liu
- Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Kaitong Liao
- Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Xiaohua Guo
- Department of Nephrology, Shenzhen Hospital, Southern Medical University, Shenzhen 518110, China
| | - Dong Wang
- Dermatology Hospital, Southern Medical University, Guangzhou 510091, China
- Department of Bioinformatics, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Ying Lin
- Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
| | - Zhili Rong
- Department of Nephrology, Shenzhen Hospital, Southern Medical University, Shenzhen 518110, China
- Cancer Research Institute, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
- Dermatology Hospital, Southern Medical University, Guangzhou 510091, China
- Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou 510005, China
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20
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Amberger M, Ivics Z. Latest Advances for the Sleeping Beauty Transposon System: 23 Years of Insomnia but Prettier than Ever: Refinement and Recent Innovations of the Sleeping Beauty Transposon System Enabling Novel, Nonviral Genetic Engineering Applications. Bioessays 2020; 42:e2000136. [PMID: 32939778 DOI: 10.1002/bies.202000136] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2020] [Revised: 07/29/2020] [Indexed: 12/13/2022]
Abstract
The Sleeping Beauty transposon system is a nonviral DNA transfer tool capable of efficiently mediating transposition-based, stable integration of DNA sequences of choice into eukaryotic genomes. Continuous refinements of the system, including the emergence of hyperactive transposase mutants and novel approaches in vectorology, greatly improve upon transposition efficiency rivaling viral-vector-based methods for stable gene insertion. Current developments, such as reversible transgenesis and proof-of-concept RNA-guided transposition, further expand on possible applications in the future. In addition, innate advantages such as lack of preferential integration into genes reduce insertional mutagenesis-related safety concerns while comparably low manufacturing costs enable widespread implementation. Accordingly, the system is recognized as a powerful and versatile tool for genetic engineering and is playing a central role in an ever-expanding number of gene and cell therapy clinical trials with the potential to become a key technology to meet the growing demand for advanced therapy medicinal products.
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Affiliation(s)
- Maximilian Amberger
- Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, D-63225, Germany
| | - Zoltán Ivics
- Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, D-63225, Germany
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21
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Chaudhari N, Rickard AM, Roy S, Dröge P, Makhija H. A non-viral genome editing platform for site-specific insertion of large transgenes. Stem Cell Res Ther 2020; 11:380. [PMID: 32883366 PMCID: PMC7650303 DOI: 10.1186/s13287-020-01890-6] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2020] [Revised: 07/22/2020] [Accepted: 08/18/2020] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND The precise, functional and safe insertion of large DNA payloads into host genomes offers versatility in downstream genetic engineering-associated applications, spanning cell and gene therapies, therapeutic protein production, high-throughput cell-based drug screening and reporter cell lines amongst others. Employing viral- and non-viral-based genome engineering tools to achieve specific insertion of large DNA-despite being successful in E. coli and animal models-still pose challenges in the human system. In this study, we demonstrate the applicability of our lambda integrase-based genome insertion tool for human cell and gene therapy applications that require insertions of large functional genes, as exemplified by the integration of a functional copy of the F8 gene and a Double Homeobox Protein 4 (DUX4)-based reporter cassette for potential hemophilia A gene therapy and facioscapulohumeral muscular dystrophy (FSHD)-based high-throughput drug screening purposes, respectively. Thus, we present a non-viral genome insertion tool for safe and functional delivery of large seamless DNA cargo into the human genome that can enable novel designer cell-based therapies. METHODS Previously, we have demonstrated the utility of our phage λ-integrase platform to generate seamless vectors and subsequently achieve functional integration of large-sized DNA payloads at defined loci in the human genome. To further explore this tool for therapeutic applications, we used pluripotent human embryonic stem cells (hESCs) to integrate large seamless vectors comprising a 'gene of interest'. Clonal cell populations were screened for the correct integration events and further characterized by southern blotting, gene expression and protein activity assays. In the case of our hemophilia A-related study, clones were differentiated to confirm that the targeted locus is active after differentiation and actively express and secrete Factor VIII. RESULTS The two independent approaches demonstrated specific and functional insertions of a full-length blood clotting F8 expression cassette of ~ 10 kb and of a DUX4 reporter cassette of ~ 7 kb in hESCs. CONCLUSION We present a versatile tool for site-specific human genome engineering with large transgenes for cell/gene therapies and other synthetic biology and biomedical applications.
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Affiliation(s)
- Namrata Chaudhari
- School of Biological Sciences, Nanyang Technological University, Singapore, 637551, Republic of Singapore
| | - Amanda M Rickard
- Genea Biocells, 11099 North Torrey Pines Road, Suite 210, La Jolla, CA, 92037, USA
| | - Suki Roy
- School of Biological Sciences, Nanyang Technological University, Singapore, 637551, Republic of Singapore
| | - Peter Dröge
- School of Biological Sciences, Nanyang Technological University, Singapore, 637551, Republic of Singapore.
| | - Harshyaa Makhija
- School of Biological Sciences, Nanyang Technological University, Singapore, 637551, Republic of Singapore.
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22
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Kumar D, Anand T, Talluri TR, Kues WA. Potential of transposon-mediated cellular reprogramming towards cell-based therapies. World J Stem Cells 2020; 12:527-544. [PMID: 32843912 PMCID: PMC7415244 DOI: 10.4252/wjsc.v12.i7.527] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/26/2020] [Revised: 05/09/2020] [Accepted: 05/28/2020] [Indexed: 02/07/2023] Open
Abstract
Induced pluripotent stem (iPS) cells present a seminal discovery in cell biology and promise to support innovative treatments of so far incurable diseases. To translate iPS technology into clinical trials, the safety and stability of these reprogrammed cells needs to be shown. In recent years, different non-viral transposon systems have been developed for the induction of cellular pluripotency, and for the directed differentiation into desired cell types. In this review, we summarize the current state of the art of different transposon systems in iPS-based cell therapies.
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Affiliation(s)
- Dharmendra Kumar
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar 125001, India.
| | - Taruna Anand
- NCVTC, ICAR-National Research Centre on Equines, Hisar 125001, India
| | - Thirumala R Talluri
- Equine Production Campus, ICAR-National Research Centre on Equines, Bikaner 334001, India
| | - Wilfried A Kues
- Friedrich-Loeffler-Institut, Institute of Farm Animal Genetics, Department of Biotechnology, Mariensee 31535, Germany
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23
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Structural basis of seamless excision and specific targeting by piggyBac transposase. Nat Commun 2020; 11:3446. [PMID: 32651359 PMCID: PMC7351741 DOI: 10.1038/s41467-020-17128-1] [Citation(s) in RCA: 44] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2020] [Accepted: 06/02/2020] [Indexed: 12/16/2022] Open
Abstract
The piggyBac DNA transposon is used widely in genome engineering applications. Unlike other transposons, its excision site can be precisely repaired without leaving footprints and it integrates specifically at TTAA tetranucleotides. We present cryo-EM structures of piggyBac transpososomes: a synaptic complex with hairpin DNA intermediates and a strand transfer complex capturing the integration step. The results show that the excised TTAA hairpin intermediate and the TTAA target adopt essentially identical conformations, providing a mechanistic link connecting the two unique properties of piggyBac. The transposase forms an asymmetric dimer in which the two central domains synapse the ends while two C-terminal domains form a separate dimer that contacts only one transposon end. In the strand transfer structure, target DNA is severely bent and the TTAA target is unpaired. In-cell data suggest that asymmetry promotes synaptic complex formation, and modifying ends with additional transposase binding sites stimulates activity.
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24
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Kovač A, Miskey C, Menzel M, Grueso E, Gogol-Döring A, Ivics Z. RNA-guided retargeting of S leeping Beauty transposition in human cells. eLife 2020; 9:e53868. [PMID: 32142408 PMCID: PMC7077980 DOI: 10.7554/elife.53868] [Citation(s) in RCA: 38] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2019] [Accepted: 03/05/2020] [Indexed: 12/12/2022] Open
Abstract
An ideal tool for gene therapy would enable efficient gene integration at predetermined sites in the human genome. Here we demonstrate biased genome-wide integration of the Sleeping Beauty (SB) transposon by combining it with components of the CRISPR/Cas9 system. We provide proof-of-concept that it is possible to influence the target site selection of SB by fusing it to a catalytically inactive Cas9 (dCas9) and by providing a single guide RNA (sgRNA) against the human Alu retrotransposon. Enrichment of transposon integrations was dependent on the sgRNA, and occurred in an asymmetric pattern with a bias towards sites in a relatively narrow, 300 bp window downstream of the sgRNA targets. Our data indicate that the targeting mechanism specified by CRISPR/Cas9 forces integration into genomic regions that are otherwise poor targets for SB transposition. Future modifications of this technology may allow the development of methods for specific gene insertion for precision genetic engineering.
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Affiliation(s)
- Adrian Kovač
- Transposition and Genome Engineering, Division of Medical Biotechnology, Paul Ehrlich InstituteLangenGermany
| | - Csaba Miskey
- Transposition and Genome Engineering, Division of Medical Biotechnology, Paul Ehrlich InstituteLangenGermany
| | | | - Esther Grueso
- Transposition and Genome Engineering, Division of Medical Biotechnology, Paul Ehrlich InstituteLangenGermany
| | | | - Zoltán Ivics
- Transposition and Genome Engineering, Division of Medical Biotechnology, Paul Ehrlich InstituteLangenGermany
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25
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Abstract
Since Barbara McClintock’s groundbreaking discovery of mobile DNA sequences some 70 years ago, transposable elements have come to be recognized as important mutagenic agents impacting genome composition, genome evolution, and human health. Transposable elements are a major constituent of prokaryotic and eukaryotic genomes, and the transposition mechanisms enabling transposon proliferation over evolutionary time remain engaging topics for study, suggesting complex interactions with the host, both antagonistic and mutualistic. The impact of transposition is profound, as over 100 human heritable diseases have been attributed to transposon insertions. Transposition can be highly mutagenic, perturbing genome integrity and gene expression in a wide range of organisms. This mutagenic potential has been exploited in the laboratory, where transposons have long been utilized for phenotypic screening and the generation of defined mutant libraries. More recently, barcoding applications and methods for RNA-directed transposition are being used towards new phenotypic screens and studies relevant for gene therapy. Thus, transposable elements are significant in affecting biology both
in vivo and in the laboratory, and this review will survey advances in understanding the biological role of transposons and relevant laboratory applications of these powerful molecular tools.
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Affiliation(s)
- Anuj Kumar
- Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI, USA.,Program in Cellular and Molecular Biology, University of Michigan, Ann Arbor, MI, USA
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26
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Querques I, Mades A, Zuliani C, Miskey C, Alb M, Grueso E, Machwirth M, Rausch T, Einsele H, Ivics Z, Hudecek M, Barabas O. A highly soluble Sleeping Beauty transposase improves control of gene insertion. Nat Biotechnol 2019; 37:1502-1512. [PMID: 31685959 PMCID: PMC6894935 DOI: 10.1038/s41587-019-0291-z] [Citation(s) in RCA: 68] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2019] [Accepted: 09/13/2019] [Indexed: 12/21/2022]
Abstract
The Sleeping Beauty (SB) transposon system is an efficient non-viral gene transfer tool in mammalian cells but its broad use has been hampered by uncontrolled transposase gene activity from DNA vectors, posing a risk for genome instability, and by the inability to use transposase protein directly. Here, we used rational protein design based on the crystal structure of the hyperactive SB100X variant to create an SB transposase (hsSB) with enhanced solubility and stability. We demonstrate that hsSB can be delivered with transposon DNA to genetically modify cell lines and embryonic, hematopoietic and induced pluripotent stem cells (iPSCs), overcoming uncontrolled transposase activity. We used hsSB to generate chimeric antigen receptor (CAR) T-cells, which exhibit potent anti-tumor activity in vitro and in xenograft mice. We found that hsSB spontaneously penetrates cells, enabling modification of iPSCs and generation of CAR-T cells without the use of transfection reagents. Titration of hsSB to modulate genomic integration frequency achieved as few as two integrations per genome.
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Affiliation(s)
- Irma Querques
- Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
| | - Andreas Mades
- Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany
| | - Cecilia Zuliani
- Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
| | - Csaba Miskey
- Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany
| | - Miriam Alb
- Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany
| | - Esther Grueso
- Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany
| | - Markus Machwirth
- Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany
| | - Tobias Rausch
- Genomics Core Facility, European Molecular Biology Laboratory, Heidelberg, Germany
| | - Hermann Einsele
- Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany
| | - Zoltán Ivics
- Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany
| | - Michael Hudecek
- Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Würzburg, Germany.
| | - Orsolya Barabas
- Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
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Wilson MH, Veach RA, Luo W, Welch RC, Roy S, Fissell WH. Genome Engineering Renal Epithelial Cells for Enhanced Volume Transport Function. Cell Mol Bioeng 2019; 13:17-26. [PMID: 32030105 DOI: 10.1007/s12195-019-00601-3] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2019] [Accepted: 09/14/2019] [Indexed: 12/20/2022] Open
Abstract
Introduction Bioengineering an implantable artificial kidney (IAK) will require renal epithelial cells capable of reabsorption of salt and water. We used genome engineering to modify cells for improved Na+/H+ exchange and H2O reabsorption. The non-viral piggyBac transposon system enables genome engineering cells to stably overexpress one or more transgenes simultaneously. Methods We generated epitope-tagged human sodium hydrogen exchanger 3 (NHE3) and aquaporin-1 (AQP1) cDNA expressing piggyBac transposon vectors. Transgene expression was evaluated via western blot and immunofluorescence. Flow cytometry analysis was used to quantitate transporter expression in a library of genome engineered clones. Cell surface biotinylation was used evaluate surface protein localization. Blister formation assays were used to monitor cellular volumetric transport. Results piggyBac enabled stable transposon integration and overexpression of cumate-inducible NHE3 and/or constitutively expressing AQP1 in cultured renal (MDCK) epithelial cells. Cell surface delivery of NHE3 and AQP1 was confirmed using cell surface biotinylation assays. Flow cytometry of a library of MDCK clones revealed varying expression of AQP1 and NHE3. MDCK cells expressing AQP1 and cumate-inducible NHE3 demonstrated increased volumetric transport. Conclusions Our results demonstrate that renal epithelial cells an be genome engineered for enhanced volumetric transport that will be needed for an IAK device. Our results lay the foundation for future studies of genome engineering human kidney cells for renal tubule cell therapy.
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Affiliation(s)
- Matthew H Wilson
- Division of Nephrology and Hypertension, Department of Medicine, Vanderbilt University Medical Center, 1161 21st Ave South, S-3223 MCN, Nashville, TN 37232 USA
- Department of Veterans Affairs, Tennessee Valley Health Services, Nashville, TN 37212 USA
- Department of Pharmacology, Vanderbilt University, Nashville, TN 37232 USA
| | - Ruth Ann Veach
- Division of Nephrology and Hypertension, Department of Medicine, Vanderbilt University Medical Center, 1161 21st Ave South, S-3223 MCN, Nashville, TN 37232 USA
| | - Wentian Luo
- Division of Nephrology and Hypertension, Department of Medicine, Vanderbilt University Medical Center, 1161 21st Ave South, S-3223 MCN, Nashville, TN 37232 USA
| | - Richard C Welch
- Division of Nephrology and Hypertension, Department of Medicine, Vanderbilt University Medical Center, 1161 21st Ave South, S-3223 MCN, Nashville, TN 37232 USA
| | - Shuvo Roy
- Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, CA 94158 USA
| | - William H Fissell
- Division of Nephrology and Hypertension, Department of Medicine, Vanderbilt University Medical Center, 1161 21st Ave South, S-3223 MCN, Nashville, TN 37232 USA
- Department of Biomedical Engineering, Vanderbilt University, Nashville, TN 37232 USA
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