For mammalian synthetic biology research, multiple orthogonal and tunable gene expression systems have been developed, among which the tetracycline (Tet)-inducible system is a key tool for gain-of-function mutations. Precise and long-lasting regulation of genetic circuits is necessary for the effective use of these systems in genetically engineered stable cell lines. However, current cell line development strategies, which depend on either random or site-specific integration along with antibiotic selection, are unpredictable and unsustainable, limiting their widespread use. To overcome these issues, we aimed to establish a Robust Overexpression via Site-specific integration of Effector (ROSE) system, a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated streamlined Tet-On3G-inducible master cell line (MCL) development platform. ROSE MCLs equipped with a landing pad facilitated the transcriptional regulation of various effector genes via recombinase-mediated cassette exchange. Long-term investigation revealed that the modular design of genetic payloads and integration sites significantly affected the induction capacity and stability, with ROSE MCLs exhibiting exceptional induction performance. To demonstrate the versatility of our platform, we explored its efficiency for the precise regulation of selection stringency, manufacturing of therapeutic antibodies with tunable expression levels and timing, and transcription factor engineering. Overall, this study demonstrated the effectiveness and reliability of the ROSE platform, highlighting its potential for various biological and biotechnological applications.

中文翻译：

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精确且可持续的多西环素诱导型细胞系开发平台，用于具有功能获得性突变的可靠哺乳动物细胞工程


对于哺乳动物合成生物学研究，已经开发了多种正交和可调基因表达系统，其中四环素 （Tet） 诱导系统是功能获得性突变的关键工具。遗传回路的精确和持久调节对于在基因工程稳定细胞系中有效使用这些系统是必要的。然而，当前的细胞系开发策略依赖于随机或位点特异性整合以及抗生素选择，是不可预测且不可持续的，限制了它们的广泛使用。为了克服这些问题，我们的目标是通过效应器的位点特异性整合 （ROSE） 系统建立稳健过表达，这是一个成簇的规则间隔短回文重复序列 （CRISPR）/CRISPR 相关蛋白 9 介导的简化 Tet-On3G 诱导型主细胞系 （MCL） 开发平台。配备着陆垫的 ROSE MCL 通过重组酶介导的盒交换促进了各种效应基因的转录调控。长期调查显示，遗传有效载荷和整合位点的模块化设计显著影响了诱导能力和稳定性，ROSE MCLs 表现出优异的诱导性能。为了证明我们平台的多功能性，我们探索了其精确调节选择严格性、生产表达水平和时间可调的治疗性抗体以及转录因子工程的效率。总体而言，这项研究证明了 ROSE 平台的有效性和可靠性，突出了其在各种生物和生物技术应用方面的潜力。