An efficient Screening System in Yeast to Select a Hyperactive piggyBac Transposase for Mammalian Applications

An efficient Screening System in Yeast to Select a Hyperactive piggyBac Transposase for Mammalian Applications

As non-viral transgenic vectors, the <i>piggyBac</i> transposon system represents an attractive tool for gene delivery to achieve a long-term gene expression in immunotherapy applications due to its large cargo capacity, its lack of a trace of transposon and of genotoxic potential, and i...

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Main Authors: Wen Wen, Shanshan Song, Yuchun Han, Haibin Chen, Xiangzhen Liu, Qijun Qian
Format: Article
Language:English
Published: MDPI AG 2020-04-01
Series:International Journal of Molecular Sciences
Subjects:
non-viral transgenic vectors
piggyBac transposase
yeast
efficient screening system
transposition efficiency
Online Access:https://www.mdpi.com/1422-0067/21/9/3064
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spelling doaj-b51460e1cd624af8bb3c2cbbd9cbfeeb2020-11-25T03:09:13ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672020-04-01213064306410.3390/ijms21093064An efficient Screening System in Yeast to Select a Hyperactive piggyBac Transposase for Mammalian ApplicationsWen Wen0Shanshan Song1Yuchun Han2Haibin Chen3Xiangzhen Liu4Qijun Qian5Shanghai Cell Therapy Research Institute, Shanghai Cell Therapy Group, Shanghai 201805, ChinaShanghai Cell Therapy Research Institute, Shanghai Cell Therapy Group, Shanghai 201805, ChinaShanghai Cell Therapy Research Institute, Shanghai Cell Therapy Group, Shanghai 201805, ChinaShanghai Cell Therapy Research Institute, Shanghai Cell Therapy Group, Shanghai 201805, ChinaShanghai Cell Therapy Research Institute, Shanghai Cell Therapy Group, Shanghai 201805, ChinaShanghai Cell Therapy Research Institute, Shanghai Cell Therapy Group, Shanghai 201805, ChinaAs non-viral transgenic vectors, the <i>piggyBac</i> transposon system represents an attractive tool for gene delivery to achieve a long-term gene expression in immunotherapy applications due to its large cargo capacity, its lack of a trace of transposon and of genotoxic potential, and its highly engineered structure. However, further improvements in transpose activity are required for industrialization and clinical applications. Herein, we established a one-plasmid effective screening system and a two-step high-throughput screening process in yeast to isolate hyperactive mutants for mammalian cell applications. By applying this screening system, 15 hyperactive <i>piggyBac</i> transposases that exhibited higher transpose activity compared with optimized hyPBase in yeast and four mutants that showed higher transpose activity in mammalian cells were selected among 3000 hyPBase mutants. The most hyperactive transposase, bz-hyPBase, with four mutation sites showed an ability to yield high-efficiency editing in Chinese hamster ovarian carcinoma (CHO) cells and T cells, indicating that they could be expanded for gene therapy approaches. Finally, we tested the potential of this screening system in other versions of <i>piggyBac</i> transposase.https://www.mdpi.com/1422-0067/21/9/3064non-viral transgenic vectorspiggyBac transposaseyeastefficient screening systemtransposition efficiency
collection DOAJ
language English
format Article
sources DOAJ
author Wen Wen
Shanshan Song
Yuchun Han
Haibin Chen
Xiangzhen Liu
Qijun Qian
spellingShingle Wen Wen
Shanshan Song
Yuchun Han
Haibin Chen
Xiangzhen Liu
Qijun Qian
An efficient Screening System in Yeast to Select a Hyperactive piggyBac Transposase for Mammalian Applications
International Journal of Molecular Sciences
non-viral transgenic vectors
piggyBac transposase
yeast
efficient screening system
transposition efficiency
author_facet Wen Wen
Shanshan Song
Yuchun Han
Haibin Chen
Xiangzhen Liu
Qijun Qian
author_sort Wen Wen
title An efficient Screening System in Yeast to Select a Hyperactive piggyBac Transposase for Mammalian Applications
title_short An efficient Screening System in Yeast to Select a Hyperactive piggyBac Transposase for Mammalian Applications
title_full An efficient Screening System in Yeast to Select a Hyperactive piggyBac Transposase for Mammalian Applications
title_fullStr An efficient Screening System in Yeast to Select a Hyperactive piggyBac Transposase for Mammalian Applications
title_full_unstemmed An efficient Screening System in Yeast to Select a Hyperactive piggyBac Transposase for Mammalian Applications
title_sort efficient screening system in yeast to select a hyperactive piggybac transposase for mammalian applications
publisher MDPI AG
series International Journal of Molecular Sciences
issn 1661-6596
1422-0067
publishDate 2020-04-01
description As non-viral transgenic vectors, the <i>piggyBac</i> transposon system represents an attractive tool for gene delivery to achieve a long-term gene expression in immunotherapy applications due to its large cargo capacity, its lack of a trace of transposon and of genotoxic potential, and its highly engineered structure. However, further improvements in transpose activity are required for industrialization and clinical applications. Herein, we established a one-plasmid effective screening system and a two-step high-throughput screening process in yeast to isolate hyperactive mutants for mammalian cell applications. By applying this screening system, 15 hyperactive <i>piggyBac</i> transposases that exhibited higher transpose activity compared with optimized hyPBase in yeast and four mutants that showed higher transpose activity in mammalian cells were selected among 3000 hyPBase mutants. The most hyperactive transposase, bz-hyPBase, with four mutation sites showed an ability to yield high-efficiency editing in Chinese hamster ovarian carcinoma (CHO) cells and T cells, indicating that they could be expanded for gene therapy approaches. Finally, we tested the potential of this screening system in other versions of <i>piggyBac</i> transposase.
topic non-viral transgenic vectors
piggyBac transposase
yeast
efficient screening system
transposition efficiency
url https://www.mdpi.com/1422-0067/21/9/3064
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