Cell lines used in bioproduction are routinely engineered to improve their production efficiency. Numerous strategies, such as random mutagenesis, RNA interference screens, and transcriptome analyses have been employed to identify effective engineering targets. A genome‐wide small interfering RNA screen previously identified the CASP8AP2 gene as a potential engineering target for improved expression of recombinant protein in the HEK293 cell line. Here, we validate the CASP8AP2 gene as an engineering target in HEK293 cells by knocking it out using CRISPR/Cas9 genome editing and assessing the effect of its knockout on recombinant protein expression, cell growth, cell viability, and overall gene expression. HEK293 cells lacking CASP8AP2 showed a seven‐fold increase in specific expression of recombinant luciferase and a 2.5‐fold increase in specific expression of recombinant SEAP, without significantly affecting cell growth and viability. Transcriptome analysis revealed that the deregulation of the cell cycle, specifically the upregulation of the cyclin‐dependent kinase inhibitor 2A (CDKN2A) gene, contributed to the improvement in recombinant protein expression in CASP8AP2 deficient cells. The results validate the CASP8AP2 gene is a viable engineering target for improved recombinant protein expression in the HEK293 cell line.

中文翻译：

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通过上调细胞周期蛋白依赖性激酶抑制剂 2A 基因，敲除 caspase 8 相关蛋白 2 基因可改善 HEK293 细胞中重组蛋白的表达。

生物生产中使用的细胞系经过常规工程改造以提高其生产效率。许多策略，如随机诱变、RNA 干扰筛选和转录组分析已被用于识别有效的工程目标。全基因组小干扰 RNA 筛选先前将CASP8AP2基因确定为改善 HEK293 细胞系中重组蛋白表达的潜在工程目标。在这里，我们通过使用 CRISPR/Cas9 基因组编辑将 CASP8AP2 基因敲除并评估其敲除对重组蛋白表达、细胞生长、细胞活力和整体基因表达的影响，验证了CASP8AP2基因作为 HEK293 细胞中的工程靶点。缺乏CASP8AP2 的HEK293 细胞显示重组荧光素酶的特异性表达增加了 7 倍，重组 SEAP 的特异性表达增加了 2.5 倍，而对细胞生长和活力没有显着影响。转录组分析表明，细胞周期的失调，特别是细胞周期蛋白依赖性激酶抑制剂 2A ( CDKN2A ) 基因的上调，有助于CASP8AP2缺陷细胞中重组蛋白表达的改善。结果验证了CASP8AP2基因是一个可行的工程靶标，可用于改善 HEK293 细胞系中的重组蛋白表达。