Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #04-01 Centros, Singapore, 138668, Singapore.
Cell Line Development, MRL, Merck & Co., Inc., 2000 Galloping Hill Road, Kenilworth, NJ, 07033, USA.
Biotechnol J. 2021 Mar;16(3):e2000267. doi: 10.1002/biot.202000267. Epub 2020 Nov 9.
The Chinese hamster ovary (CHO) cell line is commonly used for the production of biotherapeutics. As cell productivity directly affects the cost of production, methods are developed to manipulate the expression of specific genes that are known to be involved in protein synthesis, folding, and secretion to increase productivity. However, there are no large-scale CHO-specific functional screens to identify novel gene targets that impact the production of secreted recombinant proteins. Here, a large-scale, CHO cell-specific small interfering RNA screen is performed to identify genes that consistently enhance antibody production when silenced in a panel of seven CHO cell lines. Four genes, namely, Cyp1a2, Atp5s, Dgki, and P3h2, are identified, and then selected for CRISPR-Cas9 knockout validation in recombinant CHO cell lines. Single knockout of Cyp1a2, Atp5s, or Dgki, but not P3h2, results in a more than 90% increase in specific antibody productivity. Overall, the knockout of Cyp1a2 demonstrates the most significant improvement of antibody production, with a minimal impact on cell growth.
中国仓鼠卵巢（CHO）细胞系常用于生物治疗药物的生产。由于细胞产率直接影响生产成本，因此开发了各种方法来操纵已知参与蛋白质合成、折叠和分泌的特定基因的表达，以提高产率。然而，目前还没有大规模的 CHO 特异性功能筛选方法来鉴定影响分泌型重组蛋白生产的新基因靶标。在这里，进行了大规模的 CHO 细胞特异性小干扰 RNA 筛选，以鉴定在 7 种 CHO 细胞系的细胞系中沉默时能一致提高抗体产量的基因。鉴定到 Cyp1a2、Atp5s、Dgki 和 P3h2 这 4 个基因，然后选择用于重组 CHO 细胞系的 CRISPR-Cas9 敲除验证。Cyp1a2、Atp5s 或 Dgki 的单基因敲除，但不是 P3h2，会导致特定抗体产率增加超过 90%。总体而言，Cyp1a2 的敲除显示出对抗体生产的最大改善，对细胞生长的影响最小。
