Base editing-mediated exon-specific knockout of glutamine synthetase generates a high-productivity CHO cell line for antibody manufacturing.

The Chinese hamster ovary (CHO) cell platform dominates recombinant protein production, with the glutamine synthetase (GS) system widely used for clonal selection. However, we observed variable performance between two commercial CHO lines (ATCC CHO-K1 and CHOZN®), which carry distinct intragenic GS deletions and differential residual transcript levels, raising the possibility that the premature termination codon (PTC) position contributes to this phenotypic divergence. To systematically assess the impact of the PTC position, we used base editing to introduce PTCs into each of the six coding exons of Glul. Among the resulting clonal lines, the exon 7-edited (E7) variant exhibited complete GS ablation and sustained high viability. In fed-batch production of an anti-PD-1 monoclonal antibody, E7 achieved higher titers and specific productivities than CHOZN®, with sustained viability. Our findings demonstrate that PTC location is a key determinant of GS knockout completeness and cellular phenotype, identifying exon 7 as a critical knockout target that offers a defined, exon-resolved strategy for accelerating CHO cell line development and improving recombinant protein titers.