Gene editing in mammalian cells results in transient DNA breakage and replication stress

Gene editing utilizing short, single-stranded oligonucleotides (ODNs) arose as an alternative strategy to viral-based gene therapy methods. ODN-induced gene editing allows for correction of endogenous genes, maintaining desired gene expression levels and patterns, whereas viral gene therapy methods do not maintain endogenous expression levels or patterns. Viral gene therapy trials have been marred by safety concerns owing to both the virus itself and the location of gene insertion in the genome. ODN-induced gene editing is associated with the activation of many DNA damage response proteins, raising concern over possible genome-wide off-target effects. Until now, an assessment of such possible DNA breakage events has not been performed.;This dissertation demonstrates that ODN-induced gene editing causes transient double-strand break (DSB) formation related to the editing reaction in the HCT116-19 cell model system with a single-copy mutant eGFP reporter gene. The DNA breakage appears to exhibit specificity for ODNs designed to target and bind the intended genomic site. No significant off-target DNA breakage effects are detected. Additionally, DNA breakage induced by the gene editing reaction is found in S phase cells, wherein replication would be taking place.;The mechanism of ODN-induced gene editing has been heavily investigated and it is now known that replication plays a major role in the gene editing reaction. DNA damage in the context of S phase supports a model of ODN-induced replication stress. Protein alterations associated with replication stress such as Bloom's syndrome helicase accumulation, the mono-ubiquitylation of proliferating cell nuclear antigen (PCNA), and the increased association of the error-prone Y-family DNA polymerase eta with PCNA are all described herein in response to ODN-induced gene editing. This dissertation supports a model of gene editing wherein the ODN aligns in homologous register with its target genome site in the context of replication, creates replication stress by inhibiting polymerase progression, induces a DSB as part of replication fork restart, and utilizes translesion synthesis (TLS) to incorporate the ODN into the replicated strand. The resulting mismatch may be subsequently repaired or persist until another round of replication resolves the mismatch, potentially producing corrected cells. Future investigations will further elucidate the role of DSB formation and TLS in ODN-induced gene editing.