Abstract The GAPDH locus is attractive for recombinant protein expression according to knockdown and transcriptomic data as it is the 3rd most expressed gene in HEK293. DHFR KO cells growing exclusively in media supplemented in hypoxanthine and thymidine (HT). Integration of exogenous DHFR alongside the gene of interest in DHFR KO cells effectively rescues cells allowing selection simply using media without HT supplementation. CRISPR-Cas9 is an efficient gene-editing technology that allows scientists to effectively remove, insert and modify DNA in cells Introduction This proposed methodology aims to optimize recombinant protein production in HEK293 cells to improve their efficacy, lower the potential of immunogenicity and increase the industrial potential of human derived cell hosts. Significance Methods Results Conclusions Fig. 1 Bienzymatic (EcoRV & BbsI) Digestion of cloned PX330 CRISPR plasmids confirms successful guide integration Successful integration results in one band whereas unsuccessful integration results in 2 bands. For each guide, 2 clones were tested via this method and sequenced. At least one clone for each guide was successfully integrated (C, control (undigested) & D, digested) Fig. 2 PCR Screening Confirms homozygous DHFR1 Knockout Genomic DNA from single cell clones was isolated and a PCR reaction was run using primers designed outside of the determined target sites to test for a reduction in the produced fragment. All clones except clone 20 exhibit successful DHFR1 homozygous knockout. Fig. 3 Fluorescent Microscopy Confirms Stable integration of eGFP and Puromycin in HEK293 cells HEK293 WT cells were transfected with the Cas9-AAVS1 construct and the HDR-AAVS1. Cells underwent selection for 10 days using 1.5ug/ml of puromycin followed by single cell cloning to confirm stable integration of the HDR template into the AAVS1 site All CRISPR-Cas9 constructs were successfully cloned into the PX330 plasmid as confirmed by digestion using EcoRV and BbsI restriction enzymes and sequencing PCR screening shows successful isolation of monoclonal populations with desired homozygous DHFR1 knockout as evidenced by the reduction in the product size eGFP-Puro-AAVS1 cell line was successfully created as cells were selectable using puromycin and maintained eGFP expression for over 18 days post transfection Generate DHFR2 KO cells using successful DHFR1 KO clones followed by partial integration of DHFR and finally eGFP and the remaining portion of DHFR to create the complete experimental cell line. eGFP expression to be compared to that of the eGFP-Puro-AAVS1 Control Cell line using Western Blot and Flow Cytometry Confirm site specific integration of eGFP and puromycin genes in the eGFP-Puro-AAVS1 cell line by screening single cell clones using PCR and sequencing Adapt this system to produce a therapeutic recombinant protein (e.g. EPO) by cloning in the gene into the position of eGFP in the HDR-GAPDH2 plasmid Future Directions Acknowledgments I would like to thank Dr. Ihab Younis, Dr. Ravichandra Bachu and Dr. Mazen Sidani for their mentorship throughout this project. I would also like to thank Dr. Simon Faulkner and Dr. Mohamed Bouaouina for their useful insight. Finally, I would like to thank Maya Kamaldean, Bernadette Bernales and Maria Navarro for their support in the lab. References Establishing a Stable Recombinant Protein Production Cell Line Using CRISPR- Cas9 Mediated Genome Editing of HEK293 Cells Martin Sikhondze | Dr. Ravichandra Bachu | Dr. Ihab Younis | Dr. Mazen Sidani Biological Sciences Program | Carnegie Mellon University Qatar Recombinant proteins are biopharmaceuticals used in the treatment of various diseases including cancer and diabetes. Current methods to produce these therapeutics are dependent on bacterial & CHO cells however these cell hosts have non-identical post transcriptional and translation modifications increasing the likelihood of ineffective protein products in addition to possible immunoge