earticle

영어

Sialic acids present on non-reducing terminal of glycan is important for prolonged in vivo half-life of glycoproteins. Many efforts have been made to increase the sialic acid content in the manufacturing of therapeutic glycoproteins because glycoproteins containing non-sialylated glycans do not show effective in vivo efficacies owing to a short half-life. It was reported that knock down of neuraminidase expression using the RNA interference (RNAi) technology enhanced protein sialylation in CHO cells which are the most widely used for therapeutic glycoprotein production [1]. However, knock down strategy did not completely shut down the neuraminidase expression. In this study, we performed knockout of neuraminidase genes in CHO cells by using the CRISPR/Cas9 genome editing technology. Only three neuraminidase (Neu1, Neu2, and Neu3) were actively expressed in CHO cells when analyzing their expressions using real-time polymerase chain reaction (PCR). Neu3 is located in the plasma membrane while Neu1 and Neu2 exist in lysosome and cytosol, respectively. After the transfection of vectors expressing Cas9 and sgRNAs, Neu-disrupted clones were selected by using the T7 endonucleases 1 (T7E1) assay and further confirmed by the sequencing analysis of the corresponding editing sites. All of single mutants (Neu1-, Neu2-, and Neu3-disrupted clones) displayed similar proliferation ability compared to wild-type CHO cells. Two of double Neu-disrupted mutants (Neu1Neu3- and Neu2Neu3-dirupted mutants) were currently constructed. Our goal is to construct the triple knockout CHO cell mutant (Neu1Neu2Neu3-disrupted clone) and investigate its phenotypes and features.