A CRISPR/Cas9 based engineering strategy for overexpression of multiple genes in Chinese hamster ovary cells

Metabolic Engineering
Peter EisenhutNicole Borth

Abstract

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 ...Continue Reading

Citations

Aug 11, 2018·Reviews in Medical Virology·Garry A Luke, Martin D Ryan
Nov 22, 2019·Journal of Biotechnology·Mehdi Banan
Jan 12, 2021·Briefings in Bioinformatics·Hui PengJinyan Li

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