Introduction
Biopharmaceuticals have revolutionized the treatment of a wide range of diseases, and are increasingly being used in medicines (Kesik-Brodacka, 2018). Chinese hamster ovary (CHO) cells have been the most common hosts for the production of therapeutic proteins (Lai, Yang, & Ng, 2013). Traditional cell line development in CHO cells is based on random integration of recombinant constructs into the genome, resulting in clones with variable expression (Lai et al., 2013). Heterologous expression within cell pools requires the selection of multiple clones and then following a tedious screening procedure (Li, Vijayasankaran, Shen, Kiss, & Amanullah, 2010). Moreover, due to lack of control over the genome integration site, protein productivity of selected clones may decrease over time, leading to clonal heterogeneity (Coates et al., 2005). To overcome such possible shortcomings, site-specific integration (SSI) has been proposed for use in cell line development (N. K. Hamaker & K. H. Lee, 2018). SSI integrates gene-of-interest (GOI) into a predetermined genomic hotspot to enable stable and robust transgene expression. This makes it possible to generate isogenic clones with consistent productivity and stability, consequently reducing clone screening and selection time (N. K. Hamaker & K. H. Lee, 2018).
Recombinase-mediated cassette exchange (RMCE) is a procedure in reverse genetics allowing precise integration of a genomic target cassette (Turan, Zehe, Kuehle, Qiao, & Bode, 2013). Specifically, it allows double-reciprocal crossover events between two pairs of heterospecific recombinase target sites using a site-specific recombinase including Tyr-recombinases (e.g. Cre or Flp) and Ser-recombinases (e.g. ΦC31 or Bxb1) (Turan et al., 2013).Thus, RMCE exchanges only a preexisting gene cassette with an analogous one carrying GOI without changing cellular characteristics (Turan et al., 2013). It is the most advanced platform to establish new clones with similar productivity and characteristics compared to existing clones (Turan et al., 2013). Cre/Lox-based RMCE functions exceptionally well, exhibiting cassette exchange events with high fidelity (Gidoni, Srivastava, & Carmi, 2008; Schnütgen, Stewart, von Melchner, & Anastassiadis, 2006). Cre recombinase allows for repetitive cumulative gene targeting to predefined genomic loci in mammalian cells using different target sites such as Lox5171 and LoxP (Gidoni et al., 2008; Schnütgen et al., 2006).
In this study, we aimed to evaluate whether the Cre/Lox-based RMCE constitutes an effective strategy to acquire clones from existing ones established by CRISPR/Cas9-mediated SSI at a genomic hotspot allowing stable expression. Here, we report that Cre/Lox-based RMCE generates new clones by exchanging only a existing cassette with a targeting cassette carrying GOI. Our results will open up a new paradigm of commercial protein production by repeatedly establishing cell lines with predictable productivity and properties.