Chinese hamster ovary (CHO) cells continue to be the biomanufacturing platform of choice for the production of therapeutic glycoproteins, including monoclonal antibodies. The traditional cell line development workflow involves random integration of the transgene encoding the recombinant protein of interest. Random integration inherently leads to high levels of clonal heterogeneity and necessitates the tedious screening of multiple clones to isolate those with suitable performance characteristics. In contrast, site-specific integration (SSI) results in transgene knock-in events at user-defined genomic target sites, including prevalidated hot spots capable of supporting highly active and stable expression. SSI has therefore emerged as a powerful cell line engineering technique by enabling the accelerated development of production cell lines with more predictable performance and product quality. Despite these benefits, attempts to perform CRISPR-mediated SSI in CHO cells are typically met with remarkably low efficiencies, hindering efforts to isolate clones in which precise, rather than random, integration has occurred. Numerous methods have been proposed to remedy similar shortcomings observed in other mammalian cell types, yet none have been systematically evaluated in CHO. To overcome this obstacle, we developed the SSIGNAL (Site-specific Integration and GeNome ALteration) reporter system to facilitate rapid screening of CRISPR-mediated genome editing strategies. The system uses fluorescent signals to indicate genotype states, enabling data acquisition via standard flow cytometry instrumentation without the need for single-cell cloning or deep sequencing. The system was applied in CHO cells to test several current approaches for targeted integration to determine the most effective double-strand break repair strategy with the goal of improving efficiency. Ultimately, the development of robust SSI protocols may be implemented to improve long-term stability of transgenes for better cell culture process control.

More efficient site-specific integration in CHO cells enabled by a CRISPR-mediated genome editing reporter system

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.