A novel hyperactive variant of the Sleeping Beauty transposase facilitates non-viral genome engineering
The Sleeping Beauty (SB) transposon system is a useful tool for genetic applications, including gene therapy. We discovered a hyperactive variant of the SB100X transposase, called SB200X. This mutant, resulting from a specific amino acid replacement (Q124C), showed an ∼2-fold increase in transpositi...
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Elsevier
2024-12-01
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| Series: | Molecular Therapy: Nucleic Acids |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2162253124002683 |
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| author | Matthias Thomas Ochmann Csaba Miskey Lacramioara Botezatu Nicolás Sandoval-Villegas Tanja Diem Zoltán Ivics |
| author_facet | Matthias Thomas Ochmann Csaba Miskey Lacramioara Botezatu Nicolás Sandoval-Villegas Tanja Diem Zoltán Ivics |
| author_sort | Matthias Thomas Ochmann |
| collection | DOAJ |
| description | The Sleeping Beauty (SB) transposon system is a useful tool for genetic applications, including gene therapy. We discovered a hyperactive variant of the SB100X transposase, called SB200X. This mutant, resulting from a specific amino acid replacement (Q124C), showed an ∼2-fold increase in transposition activity in various human and murine cells. Other amino acid replacements in position 124 also led to a hyperactive phenotype. Position 124 is located at the very edge of the linker region that connects the DNA-binding and catalytic domains of the transposase. Consistent with a role of the linker in an autoregulatory mechanism called overproduction inhibition (OPI) in the monophyletic group of mariner transposases, we show that the hyperactivity of Q124C manifests at high concentrations of the transposase, suggesting a partial resistance of SB200X to OPI. We demonstrate that the hyperactive phenotype of Q124C can be combined with features of other useful mutations in the SB transposase. Namely, Q124C improves the transposition efficiency of the previously described K248R variant, while maintaining or even slightly improving its safer genome-wide integration profile. The SB200X transposase could enhance the utility of SB transposon-mediated genome engineering in preclinical and clinical applications. |
| format | Article |
| id | doaj-art-b005b5bfee1f43a997a1022249745dd8 |
| institution | OA Journals |
| issn | 2162-2531 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Elsevier |
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| series | Molecular Therapy: Nucleic Acids |
| spelling | doaj-art-b005b5bfee1f43a997a1022249745dd82025-08-20T02:33:08ZengElsevierMolecular Therapy: Nucleic Acids2162-25312024-12-0135410238110.1016/j.omtn.2024.102381A novel hyperactive variant of the Sleeping Beauty transposase facilitates non-viral genome engineeringMatthias Thomas Ochmann0Csaba Miskey1Lacramioara Botezatu2Nicolás Sandoval-Villegas3Tanja Diem4Zoltán Ivics5Research Center, Division of Hematology, Cell and Gene Therapy, Paul-Ehrlich-Institut, 63225 Langen, GermanyResearch Center, Division of Hematology, Cell and Gene Therapy, Paul-Ehrlich-Institut, 63225 Langen, GermanyResearch Center, Division of Hematology, Cell and Gene Therapy, Paul-Ehrlich-Institut, 63225 Langen, GermanyResearch Center, Division of Hematology, Cell and Gene Therapy, Paul-Ehrlich-Institut, 63225 Langen, GermanyResearch Center, Division of Hematology, Cell and Gene Therapy, Paul-Ehrlich-Institut, 63225 Langen, GermanyResearch Center, Division of Hematology, Cell and Gene Therapy, Paul-Ehrlich-Institut, 63225 Langen, Germany; Institute of Clinical Immunology, University of Leipzig, 04103 Leipzig, Germany; Fraunhofer Institute for Cell Therapy and Immunology, 04103 Leipzig, Germany; Corresponding author: Zoltán Ivics, Fraunhofer Institute for Cell Therapy and Immunology IZI, Perlickstrasse 1, 04103 Leipzig, Germany.The Sleeping Beauty (SB) transposon system is a useful tool for genetic applications, including gene therapy. We discovered a hyperactive variant of the SB100X transposase, called SB200X. This mutant, resulting from a specific amino acid replacement (Q124C), showed an ∼2-fold increase in transposition activity in various human and murine cells. Other amino acid replacements in position 124 also led to a hyperactive phenotype. Position 124 is located at the very edge of the linker region that connects the DNA-binding and catalytic domains of the transposase. Consistent with a role of the linker in an autoregulatory mechanism called overproduction inhibition (OPI) in the monophyletic group of mariner transposases, we show that the hyperactivity of Q124C manifests at high concentrations of the transposase, suggesting a partial resistance of SB200X to OPI. We demonstrate that the hyperactive phenotype of Q124C can be combined with features of other useful mutations in the SB transposase. Namely, Q124C improves the transposition efficiency of the previously described K248R variant, while maintaining or even slightly improving its safer genome-wide integration profile. The SB200X transposase could enhance the utility of SB transposon-mediated genome engineering in preclinical and clinical applications.http://www.sciencedirect.com/science/article/pii/S2162253124002683MT: RNA/DNA Editinggene transfernon-viral genome engineeringtransposontransposasegene insertion |
| spellingShingle | Matthias Thomas Ochmann Csaba Miskey Lacramioara Botezatu Nicolás Sandoval-Villegas Tanja Diem Zoltán Ivics A novel hyperactive variant of the Sleeping Beauty transposase facilitates non-viral genome engineering Molecular Therapy: Nucleic Acids MT: RNA/DNA Editing gene transfer non-viral genome engineering transposon transposase gene insertion |
| title | A novel hyperactive variant of the Sleeping Beauty transposase facilitates non-viral genome engineering |
| title_full | A novel hyperactive variant of the Sleeping Beauty transposase facilitates non-viral genome engineering |
| title_fullStr | A novel hyperactive variant of the Sleeping Beauty transposase facilitates non-viral genome engineering |
| title_full_unstemmed | A novel hyperactive variant of the Sleeping Beauty transposase facilitates non-viral genome engineering |
| title_short | A novel hyperactive variant of the Sleeping Beauty transposase facilitates non-viral genome engineering |
| title_sort | novel hyperactive variant of the sleeping beauty transposase facilitates non viral genome engineering |
| topic | MT: RNA/DNA Editing gene transfer non-viral genome engineering transposon transposase gene insertion |
| url | http://www.sciencedirect.com/science/article/pii/S2162253124002683 |
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