Cre/Lox-based RMCE for Site-specific Integration in CHO Cells

Traditional cell line development is based on random genomic integration of transgenes. Random integration leads to unpredictable expression and results in clonal heterogeneity requiring a tedious screening procedure. Therefore, a new strategy is needed to establish clones that exhibit stable transgene expression. Here, we performed CRISPR/Cas9-mediated site-specific integration (SSI) to incorporate a landing pad (LP; containing mCherry) at a genomic hotspot (Fer1L4) allowing stable and strong expression. Site-specific integration of LP on Fer1L4 was demonstrated by sequencing results representing the swapped sequences in mCherry-expressing cells. We then performed Cre/Lox-based recombinase-mediated cassette exchange (RMCE) to exchange LP with a targeting vector (TV; containing GFP) in clones established by CRISPR/Cas9-mediated SSI. The success of Cre/Lox-based RMCE was evidenced by sequencing results representing the swapped sequences in GFP-expressing cells. Furthermore, the swapped clones expressing GFP was enriched up to 80%, indicating that the efficiency of Cre/Lox-based RMCE would be sufficient to swap pre-existing cassettes with gene-of-interest (GOI). Taken together, our study provides a new platform for Cre/Lox-based RMCE to iteratively establish stable clones from existing ones previously established by SSI at a genomic hotspot.