Department of Breast Cancer Pathology and Research Laboratory, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin, China.
Key Laboratory of Cancer Prevention and Therapy, Tianjin, China.
Cell Mol Life Sci. 2023 Oct 10;80(11):321. doi: 10.1007/s00018-023-04976-5.
Deep mutational scanning (DMS) makes it possible to perform massively parallel quantification of the relationship between genetic variants and phenotypes of interest. However, the difficulties in introducing large variant libraries into mammalian cells greatly hinder DMS under physiological states. Here, we developed two novel strategies for DMS library construction in mammalian cells, namely 'piggyBac-in vitro ligation' and 'piggyBac-in vitro ligation-PCR'. For the first strategy, we took the 'in vitro ligation' approach to prepare high-diversity linear dsDNAs, and integrate them into the mammalian genome with a piggyBac transposon system. For the second strategy, we further added a PCR step using the in vitro ligation dsDNAs as templates, for the construction of high-content genome-integrated libraries via large-scale transfection. Both strategies could successfully establish genome-integrated EGFP-chromophore-randomized libraries in HEK293T cells and enrich the green fluorescence-chromophore amino-acid sequences. And we further identified a novel transcriptional activator peptide with the 'piggyBac-in vitro ligation-PCR' strategy. Our novel strategies greatly facilitate the construction of large variant DMS library in mammalian cells, and may have great application potential in the future.
深度突变扫描（DMS）使得对遗传变异与感兴趣表型之间的关系进行大规模并行量化成为可能。然而，在生理状态下将大型变异文库导入哺乳动物细胞中存在困难，这极大地阻碍了 DMS 的发展。在这里，我们开发了两种在哺乳动物细胞中构建 DMS 文库的新策略，即“piggyBac-体外连接”和“piggyBac-体外连接-PCR”。对于第一种策略，我们采用“体外连接”方法制备高度多样化的线性 dsDNA，并使用 piggyBac 转座子系统将其整合到哺乳动物基因组中。对于第二种策略，我们进一步添加了一个 PCR 步骤，使用体外连接 dsDNA 作为模板，通过大规模转染构建高含量的基因组整合文库。这两种策略都能够成功地在 HEK293T 细胞中建立基因组整合的 EGFP-发色团随机文库，并富集绿色荧光-发色团氨基酸序列。我们还使用“piggyBac-体外连接-PCR”策略鉴定了一种新型转录激活肽。我们的新策略极大地促进了哺乳动物细胞中大型变异 DMS 文库的构建，并且在未来可能具有很大的应用潜力。
