Characterization and mutagenesis of Chinese hamster ovary cells endogenous retroviruses to inactivate viral particle release.

The Chinese hamster ovary (CHO) cells used to produce biopharmaceutical proteins are known to contain type-C endogenous retrovirus (ERV) sequences in their genome and to release retroviral-like particles. Although evidence for their infectivity is missing, this has raised safety concerns. As the genomic origin of these particles remained unclear, we characterized type-C ERV elements at the genome, transcriptome, and viral particle RNA levels.

A role for alternative end-joining factors in homologous recombination and genome editing in Chinese hamster ovary cells.

CRISPR technologies greatly foster genome editing in mammalian cells through site-directed DNA double strand breaks (DSBs). However, precise editing outcomes, as mediated by homologous recombination (HR) repair, are typically infrequent and outnumbered by undesired genome alterations. By using knockdown and overexpression studies in Chinese hamster ovary (CHO) cells as well as characterizing repaired DNA junctions, we found that efficient HR-mediated genome editing depends on alternative end-joining (alt-EJ) DNA repair activities, a family of incompletely characterized DNA repair pathways traditionally considered to oppose HR.

MAR-Mediated transgene integration into permissive chromatin and increased expression by recombination pathway engineering.

Untargeted plasmid integration into mammalian cell genomes remains a poorly understood and inefficient process. The formation of plasmid concatemers and their genomic integration has been ascribed either to non-homologous end-joining (NHEJ) or homologous recombination (HR) DNA repair pathways. However, a direct involvement of these pathways has remained unclear.

A role for homologous recombination proteins in cell cycle regulation.

Eukaryotic cells respond to DNA breaks, especially double-stranded breaks (DSBs), by activating the DNA damage response (DDR), which encompasses DNA repair and cell cycle checkpoint signaling. The DNA damage signal is transmitted to the checkpoint machinery by a network of specialized DNA damage-recognizing and signal-transducing molecules. However, recent evidence suggests that DNA repair proteins themselves may also directly contribute to the checkpoint control.