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

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 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.

Cell Engineering and Cultivation of Chinese Hamster Ovary Cells for the Development of Orthogonal Eukaryotic Cell-free Translation Systems

“…Here we conduct an alternative approach to generate an in vitro orthogonal translation system by utilizing CHO cells stably transfected with eAzFRS. Several studies have demonstrated the use of CRISPR/Cas technology to overexpress a diverse repertoire of proteins from defined genomic loci in CHO cells ( Lee et al, 2015 ; Eisenhut et al, 2018 ; Iwao et al, 2021 ). The genomic loci HPRT1 and C12orf35 were investigated for the generation of stable CHO cell lines using CRISPR/Cas, as reported in a recent study ( Zhao et al, 2018 ).…”

Systematic use of synthetic 5′-UTR RNA structures to tune protein translation improves yield and quality of complex proteins in mammalian cell factories

“…Enhanced designs of the RgE sequences, i.e. by including CRISPR/Cas9 target sequences in the DNA ( 68 ) or N 6 -methyladenosine sites in the RNA hairpin that can selectively be modified by novel CRISPR systems ( 69 , 70 ), could be employed to delete, diversify or destabilize the RgE structure. This potentially allows to rationally vary expression levels from one initial structure.…”

“Therapeutic applications of the ‘NPGP’ family of viral 2As”

“…For mammalian cells, a novel strategy using multiplexable activation of artificially repressed genes (MAARGE) allows the fast and convenient generation of stable cell lines with defined expression of the gene(s) of interest, while avoiding off‐target activity at different sites in the genome. Since the activation of gene expression occurs at low efficiency, the expression of each engineered gene should be coupled to the expression of a distinct fluorescent protein via a 2A peptide in order to facilitate enrichment of modified cells …”