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piggyBac-system-mediated genomic integration of large variant libraries for deep mutational scanning in mammalian cells

Yanjie Zhao, Yifan Li, Yi Wang, Weijun Su, View ORCID ProfileShuai Li
doi: https://doi.org/10.1101/2022.01.17.476579
Yanjie Zhao
1Department of Breast Cancer Pathology and Research Laboratory, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin; Tianjin’s Clinical Research Center for Cancer, Tianjin 300060, China
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Yifan Li
1Department of Breast Cancer Pathology and Research Laboratory, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin; Tianjin’s Clinical Research Center for Cancer, Tianjin 300060, China
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Yi Wang
1Department of Breast Cancer Pathology and Research Laboratory, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin; Tianjin’s Clinical Research Center for Cancer, Tianjin 300060, China
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Weijun Su
2School of Medicine, Nankai University, Tianjin 300071, China
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Shuai Li
1Department of Breast Cancer Pathology and Research Laboratory, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer; Key Laboratory of Cancer Prevention and Therapy, Tianjin; Tianjin’s Clinical Research Center for Cancer, Tianjin 300060, China
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  • ORCID record for Shuai Li
  • For correspondence: shuaili@tmu.edu.cn shuai.li2001@gmail.com
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Abstract

There is a need of introducing large variant libraries into mammalian cells to facilitate deep mutational scanning (DMS). In this study, we develop an approach to integrate in-vitro ligation dsDNA variant library into cultured mammalian cell genome via the piggyBac transposon system. T4 DNA ligase was employed to produce dsDNA ligation products containing variant library sequence flanked by piggyBac inverted terminal repeat sequences (ITRs) at both ends. The ligation products were then cotransfected with a piggyBac transposase expression vector into mammalian cells for library integration. The clone formation assay results showed that about 29,000 resistance clones were generated from a single well of 6-well-plate transfected HEK293T cells. As a proof of principle of using the strategy for DMS, a GFP chromophore random variant library was integrated into the HEK293T cell genome. By FACS selection, we enriched GFP-positive cells. The next-generation sequencing (NGS) results showed that the GFP-chromophore amino acid sequences were restored after selection.

Competing Interest Statement

The authors have declared no competing interest.

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Posted January 17, 2022.
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piggyBac-system-mediated genomic integration of large variant libraries for deep mutational scanning in mammalian cells
Yanjie Zhao, Yifan Li, Yi Wang, Weijun Su, Shuai Li
bioRxiv 2022.01.17.476579; doi: https://doi.org/10.1101/2022.01.17.476579
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piggyBac-system-mediated genomic integration of large variant libraries for deep mutational scanning in mammalian cells
Yanjie Zhao, Yifan Li, Yi Wang, Weijun Su, Shuai Li
bioRxiv 2022.01.17.476579; doi: https://doi.org/10.1101/2022.01.17.476579

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