Efficient Construction of Recombinant CHO Cells for Stable Expression of Therapeutic Proteins Based on New Hotspot...

Efficient generation of stably express therapeutic recombinant proteins in Chinese hamster ovary (CHO) cell lines remains as a major challenge in biologics development. Among available strategies, CRISPR/Cas9-mediated homology-directed repair (HDR) has emerged as a highly efficient method for inserting genes of interest (GOIs) into predefined genomic 'hotspot.' In this study, we report such a 'hotspot' (LOC site) which has discovered previously by our research team, which is a non-coding RNA region within the CHO-K1 genome at position 1689 of NW_003626341.1, located in the LOC103162981 gene. Utilizing this 'hotspot,' the study implemented an integrated platform combining CRISPR/Cas9 genome editing with Bxb1 recombinase-mediated cassette exchange (RMCE) to enable efficient GOIs integration and followed by sustained expression. First, to create a fluorescent landing pad (LP) cell by inserting a Bxb1 attP site, an EGFP cassette and a promoter trap into LOC site via CRISPR/Cas9. Then, LP cell undergoes subculture for 60 consecutive generations, and the fluorescence was consistently observed, illustrating the capacity of LOC site for stable EGFP expression. Donor cassettes harboring GOIs flanked by Bxb1 attB sites were then easily swapped in through RMCE. This platform enabled one‑step insertion and stable expression of therapeutic target proteins ranging from 12 to 190 kDa including human serum albumin (HSA), interferon alpha-2b (INFα2b), recombinant factor VII (rFVII), monoclonal antibodies against the epidermal growth factor receptor (anti-EGFR, AE01), and a disintegrin and metalloproteinase with thrombospondin type 1 motif, 13 (ADAMST13). Here, we efficiently generated HSA CHO cell clones within three weeks; HSA titers reached 220 mg/L in shake‑flask batch culture conditions. Collectively, this study establishes LOC site as a 'hotspot' and shows that the CRISPR/Cas9‑Bxb1 pipeline markedly accelerates CHO cell‑line development for biologics manufacturing.