원문정보
초록
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 sialidase 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 sialidase expression. In this study, we performed knockout of sialidase genes in CHO cells by using the CRISPR/Cas9 genome editing technology. Only three sialidases (Neu1, Neu2, and Neu3) were actively expressed in CHO cells when analyzing their expressions using real-time polymerase chain reaction. First, we disrupted Neu3 gene because it 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, Neu1 gene-disrupted clones were selected by using the T7 endonucleases 1 (T7E1) assay and cell surface sialidase activity assay. Disruption of Neu1 gene did not affect the viability of CHO cells. The disruptions of Neu1 and Neu2 genes in CHO cells are currently in the progress.