Multiplex Functional Characterization of Protein Variant Libraries in Mammalian Cells with Single-Copy Genomic Integration and High-Throughput DNA Sequencing.
Multiplex Functional Characterization of Protein Variant Libraries in Mammalian Cells with Single-Copy Genomic Integration and High-Throughput DNA Sequencing.
复制标题
通过单拷贝基因组整合和高通量 DNA 测序对哺乳动物细胞中蛋白质变体文库进行多重功能表征。
DOI：
10.1007/978-1-0716-3718-0_10
复制
发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Matreyek,KennethA
中科院分区：
文献类型：
--
作者：
Kamath,NishaD;Matreyek,KennethA
基于测序的大规模并行遗传分析能够同时表征编码数千种独特蛋白质变体的文库的基因型-表型关系。由于质粒转染和慢病毒转导具有限制混合文库的特性，我们开发了一个哺乳动物合成生物学平台，利用Bxb1噬菌体DNA重组酶将编码感兴趣的转基因的单个无启动子质粒插入到细胞基因组内预先设计的“着陆点”位置。转基因在基因组整合启动子后表达，确保每个细胞只表达一个转基因，保持严格的基因型-表型联系。根据期望的表型选择细胞后，可以对转基因进行测序，以赋予每个变体表型评分。我们描述了如何创建和利用着陆垫细胞大规模，文库为基础的遗传实验。使用所提供的示例，实验模板可用于探索哺乳动物细胞内各种生物问题中的蛋白质变异。
Sequencing-based, massively parallel genetic assays have enabled simultaneous characterization of the genotype–phenotype relationships for libraries encoding thousands of unique protein variants. Since plasmid transfection and lentiviral transduction have characteristics that limit multiplexing with pooled libraries, we developed a mammalian synthetic biology platform that harnesses the Bxb1 bacteriophage DNA recombinase to insert single promoterless plasmids encoding a transgene of interest into a pre-engineered “landing pad” site within the cell genome. The transgene is expressed behind a genomically integrated promoter, ensuring only one transgene is expressed per cell, preserving a strict genotype–phenotype link. Upon selecting cells based on a desired phenotype, the transgene can be sequenced to ascribe each variant a phenotypic score. We describe how to create and utilize landing pad cells for large-scale, library-based genetic experiments. Using the provided examples, the experimental template can be adapted to explore protein variants in diverse biological problems within mammalian cells.