Knockout of cytidine monophosphate-N-acetylneuraminic acid hydroxylase in Chinese hamster ovary cells by CRISPR/Cas9-based gene-editing technology

Abstract

Chinese hamster ovary (CHO) cells are mainly used mammalian host to produce therapeutic recombinant glycoproteins which are close to the human-like glycosylation of glycoproteins. However, cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) in CHO cells can produce N-glycolylneuraminic acids (Neu5Gc) which elicits the immune response in the human body. Therefore, it is vital to silence the expression of CMAH in CHO cells using gene-editing technology. CRISPR (clustered regulatory interspaced short palindromic repeat)/Cas9-based gene editing system was performed to knock out the expression of CMAH in CHO cells. Plasmids containing Cas9, single guide (sg) RNA and reporter sequences of CMAH gene were constructed and transformed into HEK293 T cells to screen the sgRNA with high cleavage efficiency. Then the plasmids were co-transfected into the CHO cells. Eight CHO monoclonal cells were obtained by flow cytometry sorting and expanded. Base deletion mutations were identified by PCR and Sanger sequencing. qRT-PCR and MTT assay were confirmed the CMAH gene mutation in the CHO cells. Moreover, the recombinant human erythropoietin (rEPO) protein was also expressed in the mutant CHO cells, and the N-Glycosylation of rEPO showed that G0F, G1F-GN increased and G0F-GN, G0, Man5 were decreased. CRISPR/Cas9 system is a very convenient system for editing CMAH gene in CHO cells. The mutant CHO cells can be used as host to express the recombinant glycoprotein.