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While metabolic selection systems are favored over antibiotic\u2010based ones to avoid purification and safety issues, traditional single\u2010marker systems poorly regulate the light\u2010to\u2010heavy chain (LC\/HC) ratio, which is key for yield. Although novel multi\u2010marker systems can address this, they often lack strategies to enhance expression. To overcome these limitations, our study employed a triple\u2010marker 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\u2010targeting CRISPRi with a Tet\u2010on system, to enhance its efficiency. This strategy upregulated three fluorescent reporter genes, resulting in a selected mini\u2010pool with 84.84% higher cell\u2010specific productivity and 87.50% higher final titer in tyrosine\u2010free perfusion culture. Mechanistically, doxycycline (Dox)\u2010induced dCas9 expression inhibited the transcription of SV40\u2010driven 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\u2010driven systems, offering novel insights for optimizing metabolic selection.<\/jats:p>","DOI":"10.1002\/biot.70265","type":"journal-article","created":{"date-parts":[[2026,6,17]],"date-time":"2026-06-17T11:41:07Z","timestamp":1781696467000},"source":"Crossref","is-referenced-by-count":0,"title":["Attenuation of Triple Selection Markers to Enhance mAb Expression in Tyrosine\u2010Based Selection Systems via an Inducible CRISPRi Platform"],"prefix":"10.1002","volume":"21","author":[{"given":"Lei","family":"Cao","sequence":"first","affiliation":[{"name":"State Key Laboratory of Bioreactor Engineering East China University of Science and Technology  Shanghai China"},{"name":"Shanghai Collaborative Innovation Center for Biomanufacturing Technology (SCIBT)  Shanghai China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liang","family":"Zhao","sequence":"additional","affiliation":[{"name":"Shanghai Bioengine Sci\u2010Tech Co., LTD  Shanghai P. 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