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.

中文翻译：

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用于哺乳动物细胞深度突变扫描的基于线性 dsDNA 的文库的 piggyBac 介导的基因组整合

深度突变扫描 (DMS) 使得对遗传变异和感兴趣的表型之间的关系进行大规模并行量化成为可能。然而，将大型变异库引入哺乳动物细胞的困难极大地阻碍了生理状态下的 DMS。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。对于第一个策略，我们采用体外连接方法制备高多样性线性 dsDNA，并使用 piggyBac 转座子系统将它们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基因组整合文库。这两种策略都可以在 HEK293T 细胞中成功建立基因组整合的 EGFP-生色团随机文库，并丰富绿色荧光-生色团氨基酸序列。并且我们通过 piggyBac 体外连接-PCR 策略进一步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 库的构建，并且在未来可能具有巨大的应用潜力。