Establishing a Stable Recombinant Protein Production Cell Line Using CRISPR-Cas9 Mediated Genome Editing of HEK293 Cells

Recombinant proteins are effective therapeutics for many diseases and thus their demand has increased over the past few decades. Today, most recombinant proteins are produced in bacteria and non-mammalian cells hosts such as Chinese Hamster Ovary (CHO) due to their high production efficiencies. Despite this, issues pertaining to the utilization of these cell hosts include differences in post-transcriptional/translational modifications which can reduce the efficacy of produced therapeutics in addition to increasing the risk of immunogenicity. Human embryonic kidney 293 (HEK293) cells are a human-derived host system employed in the production of recombinant proteins however they are commonly used for transient expression thus demanding more research to increase their stable expression capabilities. Efficient DHFR knockout clones have been established both in CHO and HEK293 cells for recombinant protein production using the dihydrofolate reductase/methotrexate (DHFR/MTX) system which allows for selection and gene amplification. In this study, we propose a methodology to stably express recombinant proteins in HEK293s at high efficiencies. This is achieved by using CRISPR-Cas9 system to introduce DHFR and a gene of interest into one allele copy of GAPDH in DHFR double knockout HEK293s. This methodology allows for the adaptation of this expression system to produce therapeutically significant recombinant proteins.