Targeted engineering of mammalian cells has been widely attempted to ensure the efficient production of therapeutic proteins with proper quality during bioprocesses. However, the identification of novel targets for cell engineering is labor-intensive and has not yet been fully substantiated. Here, we established a CRISPR/Cas9 library screening platform in human embryonic kidney (HEK293) cells based on guide RNA integration mediated by recombinase-mediated cassette exchange (RMCE) to interrogate gene function in a high-throughput manner. This platform was further advanced using a nuclear localization signal-tagged recombinase that increased RMCE efficiency by 4.8-fold. Using this platform, we identified putative target genes, such as CDK8, GAS2L1, and GSPT1, and their perturbation confers resistance to hyperosmotic stress that inhibits cell growth and induces apoptosis. Knockout of these genes in monoclonal antibody (mAb)-producing recombinant HEK293 (rHEK293) cells enhanced resistance to hyperosmotic stress-induced apoptosis, resulting in enhanced mAb production. In particular, GSPT1-knockout yielded 2.3-fold increase in maximum mAb concentration in fed-batch culture where hyperosmotic stress naturally occurs due to nutrient feeding. Taken together, this streamlined screening platform allows the identification of novel targets associated with hyperosmotic stress, enabling the development of stress-resistant cells producing recombinant proteins.

哺乳动物细胞的靶向工程已被广泛尝试，以确保在生物过程中有效生产具有适当质量的治疗性蛋白质。然而，细胞工程新靶点的鉴定是劳动密集型的，尚未得到充分证实。在这里，我们建立了一个基于重组酶介导的盒交换（RMCE）介导的指导RNA整合的人胚胎肾（HEK293）细胞中的CRISPR/Cas9文库筛选平台，以高通量方式询问基因功能。使用核定位信号标记重组酶进一步推进了该平台，将 RMCE 效率提高了 4.8 倍。使用该平台，我们确定了推定的靶基因，例如CDK8、GAS2L1和GSPT1，并且它们的扰动赋予了对抑制细胞生长并诱导细胞凋亡的高渗应激的抵抗力。在产生单克隆抗体 (mAb) 的重组 HEK293 (rHEK293) 细胞中敲除这些基因增强了对高渗应激诱导的细胞凋亡的抵抗力，从而增强了 mAb 的产生。特别是，GSPT1基因敲除在补料分批培养中产生了 2.3 倍的最大 mAb 浓度增加，其中由于营养供给而自然发生高渗应激。总之，这个简化的筛选平台允许识别与高渗应激相关的新靶点，从而能够开发产生重组蛋白的抗应激细胞。