Correction of Mutated DNA Repair Genes as a Novel Strategy to Mitigate Genome Instability in Cell Lines Used for Therapeutic Protein Production Philipp N. Spahn1,2, Qing Hu4, Hooman Hefzi3, Shangzhong Li3, Henry Huang3, Peter Ly4 & Nathan E. Lewis1,2,3 1 Department of Pediatrics, University of California, San Diego, La Jolla, CA 92093 2 The Novo Nordisk Foundation Center for Biosustainability at UCSD, La Jolla, CA 92093 3 Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093 4 Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390 Therapeutic proteins constitute a major class of biopharmaceuticals, and include treatments for cancer, inflammation, and hereditary disorders. The overwhelming fraction of therapeutic proteins is expressed in Chinese Hamster Ovary (CHO) cells, due to their resistance to human viruses and their human-like glycosylation patterns. However, efficient high-yield protein expression remains aggravated by the instability of the CHO genome, as random chromosome rearrangements and deletions can reduce transgene copy number and lead to a loss in protein titer. So far, thorough investigations of the genetic basis of genome instability in CHO as well as effective mitigation strategies have been scarce. We analyzed whole-genome sequencing data from 11 CHO cell lines used in industrial protein production and found a high number of likely deleterious single-nucleotide polymorphisms (SNPs) in genes across all major DNA repair pathways. Using GFP reporter assays and DNA repair foci analysis, we found that double-strand-break (DSB) repair is compromised in CHO cells. Restoration of key DNA repair genes, such as ATM, PRKDC, or XRCC6, through either CRISPR/Cas9-mediated SNP reversal or overexpression of an intact wildtype copy, improved DSB repair ability, and led to a reduction of genome fragmentation, as indicated by comet assays. After culturing these DNA-repair-optimized cell lines over a period of 60 days, we analyzed chromosomal integrity by multi-color in-situ hybridization (“chromosome painting”) and found that karyotypes showed fewer structural aberrations, compared to wildtype CHO, indicating successful mitigation of genome instability. We also found that restoration of XRCC6 in a protein-producing CHO line (secreted alkaline phosphatase, SEAP) was sufficient to prevent the loss of protein titer, over a 90-day period. These results show for the first time that correction of only few DNA repair genes can results in considerable improvements in DNA repair, genome stability, and protein expression in CHO. This opens a plethora of new opportunities for the development of superior protein expression hosts. Curiously, we also find that correction of identical genes can have opposite effects in different CHO cell lines; this provides a promising model platform to study synergistic gene relationships and competition within the DSB repair hierarchy.