Chinese hamster ovary (CHO) cells are the workhorse for producing complex biopharmaceuticals. CHO cell line plasticity is advantageous to the production of exogenous proteins (<i>e.g.,</i> monoclonal antibodies) but may be a cause of cell line instability. Genome instability and the reduction of the transgene copy number decrease protein production in long-term cultures. A more stable protein production could be achieved by developing a more stable host CHO cell line.<br/>DNA double-strand breaks (DSBs) threaten genome integrity, can cause DNA mutations that reduce the production of therapeutic proteins, and can cause cell death. In immortalized CHO cell lines, certain DNA DSB repair genes are mutated compared to other mammalian organisms, including the Chinese hamster. Some CHO DNA repair genes that have single-nucleotide mutations in highly conserved regions across mammalian genomes have been identified. These single-nucleotide mutations are potential targets for genomic modification because they may have an impact on CHO cell line stability.<br/>This project aims to develop a more stable CHO cell line to produce exogenous protein in long-term cultures. This will be achieved by studying the relationship between the cell line stability and these DNA DSB repair genes. We show progress to alter CHO chromosomal genes to match the protein amino acid sequences with that of the Chinese hamster using the CRISPR-based prime-editing method. This process has been performed with multiple gene targets on two CHO cell lines. The initial attempts showed successful editing but too low of an efficiency to isolate clones. To increase the desired modification editing efficiency, we develop a two-fluorophore system to be used with fluorescence-activated cell sorting. We show higher editing efficiency can be achieved using an additional guide RNA to nick the unmodified strand. Up to 1% of desired genome modification is achieved after transfection and sorting as quantified using deep amplicon sequencing.

Progress toward a more stable host CHO cell line by restoring DNA double-strand break repair genes

ACS Spring 2022

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