ACIB Gmbh, Austrian Centre of Industrial Biotechnology, Graz, Austria; Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria.
GE Healthcare Bio-Science AB, Uppsala, Sweden.
Metab Eng. 2018 Jul;48:72-81. doi: 10.1016/j.ymben.2018.05.017. Epub 2018 May 28.
Manipulation of multiple genes to engineer Chinese Hamster Ovary (CHO) cells for better performance in production processes of biopharmaceuticals has recently become more and more popular. Yet, identification of useful genes and the unequivocally assessment of their effect alone and in combination(s) on the cellular phenotype is difficult due to high variation between subclones. Here, we present development and proof-of-concept of a novel engineering strategy using multiplexable activation of artificially repressed genes (MAARGE). This strategy will allow faster screening of overexpression of multiple genes in all possible combinations. MAARGE, in its here presented installment, comprises four different genes of interest that can all be stably integrated into the genome from one plasmid in a single transfection. Three of the genes are initially repressed by a repressor element (RE) that is integrated between promoter and translation start site. We show that an elongated 5'-UTR with an additional transcription termination (poly(A)) signal most efficiently represses protein expression. Distinct guide RNA (gRNA) targets flanking the REs for each gene then allow to specifically delete the RE by CRISPR/Cas9 and thus to activate the expression of the corresponding gene(s). We show that both individual and multiplexed activation of the genes of interest in a stably transfected CHO cell line is possible. Also, upon transfection of this stable cell line with all three gRNAs together, it was possible to isolate cells that express all potential gene combinations in a single experiment.
最近，通过操纵多个基因来设计中国仓鼠卵巢（CHO）细胞，以提高生物制药生产过程中的性能，变得越来越流行。然而，由于亚克隆之间存在高度变异性，因此识别有用的基因及其单独和组合对细胞表型的明确评估具有挑战性。在这里，我们提出了一种使用人工抑制基因的多重激活（MAARGE）的新型工程策略的开发和概念验证。该策略将允许更快地筛选所有可能组合中的多个基因的过表达。MAARGE 在其当前版本中，包含四个不同的感兴趣基因，这些基因都可以从一个质粒在单次转染中稳定整合到基因组中。这三个基因最初受到位于启动子和翻译起始位点之间的抑制元件（RE）的抑制。我们表明，具有额外转录终止（poly(A)）信号的延长 5'-UTR 最有效地抑制蛋白质表达。然后，针对每个基因的侧翼 RE 的独特向导 RNA（gRNA）靶标允许通过 CRISPR/Cas9 特异性删除 RE，从而激活相应基因的表达。我们表明，在稳定转染的 CHO 细胞系中，单个和多重激活感兴趣的基因是可能的。此外，通过共转染这一稳定细胞系中的三个 gRNA，有可能在单个实验中分离出表达所有潜在基因组合的细胞。
