Rapid development of stable transgene CHO cell lines by CRISPR/Cas9-mediated site-specific integration into C12orf35

摘要: Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production. However, by conventional random integration strategy, development of a high-expressing and stable CHO cell line has always been difficult task due to heterogenic insertion its caused requirement multiple rounds selection. Site-specific transgenes into hot spots is an ideal strategy overcome these challenges since it can generate isogenic lines with consistent productivity stability. In this study, we investigated three sites potential high transcriptional activities: C12orf35, HPRT, GRIK1, determine possible in cells, further construct reliable site-specific develop efficiently. Genes encoding representative proteins mCherry anti-PD1 monoclonal antibody were targeted loci respectively through CRISPR/Cas9 technology. Stable generated successfully after single round comparison control, all showed higher productivity, among which C12orf35 locus was advantageous both Binding affinity N-glycan analysis revealed that batches product similar quality independent on integrated sites. Deep sequencing demonstrated there low level off-target mutations CRISPR/Cas9, but none them contributed process transgene lines. Our results feasibility as target site exogenous gene integration, strongly suggested mediated rapid