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

Authors :
Wen Wen
Shanshan Song
Yuchun Han
Haibin Chen
Xiangzhen Liu
Qijun Qian
Source :
International Journal of Molecular Sciences, Vol 21, Iss 9, p 3064 (2020)
Publication Year :
2020
Publisher :
MDPI AG, 2020.

Abstract

As non-viral transgenic vectors, the piggyBac 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 piggyBac 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 piggyBac transposase.

Details

Language :
English
ISSN :
14220067 and 16616596
Volume :
21
Issue :
9
Database :
Directory of Open Access Journals
Journal :
International Journal of Molecular Sciences
Publication Type :
Academic Journal
Accession number :
edsdoj.b51460e1cd624af8bb3c2cbbd9cbfeeb
Document Type :
article
Full Text :
https://doi.org/10.3390/ijms21093064