Using CRISPR-Cas9 To Construct GNT1 Gene Knockout Cell Line

Rare diseases generally refer to diseases with low incidence and small group of patients.There is a certain type of rare diseases called lysosomal storage disorder.Glucocerebroside is a kind of familial lipid metabolism disease,which is also a rare disease.The pathogenesis of this disease is not completely clear,and the effective treatment is mainly enzyme replacement therapy.In order to improve the replacement therapy efficacy of beta glycosidase enzymes,its terminal mannose residues must be exposed outside at the end of sugar chain,so that it can bind mannose receptor in lysosomes,which can digest glycosidase enzyme substrates to achieve the goal of treatment for this disease.Beta-1,2-N-acetyl glucosamine transferase ?(GNT1)is one of the key enzymes in mammalian cells which can process glycoprotein N of sugar chain,which thus can catalyze sugar chain composed of mannose type to hybrid type transformation.The content of mannose can be increased due to GNT1 gene knockout and exposing it to the terminal of sugar chain.This research uses novel gene editing tools clustered regularly interspaced short of palindromes repeat guide nuclease repetition of RNA(CRISPR-Cas9)to edit the GNT1 gene in CHO cell.Using single guide RNA to knock out the GNT1 gene and successfully build the GNT1 knockout monoclonal cell lines,making the mannose percent of cell line expressing beta-glycosidase increase from 0.5% to 94.1% and the enzyme activity is up to 24%.High mannose type sugar content can significantly improve the beta glycosidase activity,which can further improve the efficiency when binding with mannose receptor in lysosomes,so that to improve the effect of enzyme replacement therapy efficacy.