Attenuation of Triple Selection Markers to Enhance mAb Expression in Tyrosine‐Based Selection Systems via an Inducible CRISPRi Platform

Chinese hamster ovary (CHO) cells are the predominant host for monoclonal antibody (mAb) production, where the choice of selection system is critical. While metabolic selection systems are favored over antibiotic-based ones to avoid purification and safety issues, traditional single-marker systems poorly regulate the light-to-heavy chain (LC/HC) ratio, which is key for yield. Although novel multi-marker systems can address this, they often lack strategies to enhance expression. To overcome these limitations, our study employed a triple-marker system based on the tyrosine biosynthetic pathway to regulate the LC/HC ratio and eliminate the need for alkaline concentrated tyrosine feeding. Furthermore, we introduced an inducible CRISPR interference (iCRISPRi) platform, which integrates SV40 promoter-targeting CRISPRi with a Tet-on system, to enhance its efficiency. This strategy upregulated three fluorescent reporter genes, resulting in a selected mini-pool with 84.84% higher cell-specific productivity and 87.50% higher final titer in tyrosine-free perfusion culture. Mechanistically, doxycycline (Dox)-induced dCas9 expression inhibited the transcription of SV40-driven markers, enriching cells with high marker integration under tyrosine deprivation and consequently increasing HC and LC gene copy numbers to boost mAb yields. This iCRISPRi strategy serves as a potential enhancer for SV40 promoter-driven systems, offering novel insights for optimizing metabolic selection.