DNA damage response pathway and replication fork stress during oligonucleotide directed gene editing

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• Single-stranded DNA oligonucleotides (ODNs) are tools for mammalian gene editing.
• Gene editing involves DNA repair pathways that can cause DNA breaks.
• Phosphorothioated ODNs activate DNA double-strand break markers like γH2AX.

Purpose of the Study:

• To investigate the DNA damage response to gene editing ODNs.
• To assess replication stress induced by various ODNs.
• To evaluate the impact of ODN chemical modifications on DNA damage and gene editing efficiency.

Main Methods:

• Utilized a mutant enhanced green fluorescent protein (eGFP) gene as a target for gene editing.
• Monitored DNA damage response markers, including γH2AX and modified proliferating cell nuclear antigen (PCNA).
• Compared the effects of unmodified and modified (phosphothioate) ODNs on DNA integrity and gene editing outcomes.

Main Results:

• Gene editing with ODNs activates the DNA damage response and PCNA modification, indicating replication stress.
• The gene editing process itself can cause transient DNA breaks, possibly via replication fork collapse.
• Unmodified ODNs induce less replication stress but are also less effective for gene editing compared to modified ODNs.
• Modified phosphothioate oligonucleotides (PTOs) cause significant cellular damage regardless of sequence.

Conclusions:

• Gene editing with ODNs triggers DNA damage and replication stress responses in mammalian cells.
• Chemically modified ODNs, while enhancing gene editing, induce greater collateral DNA damage.
• The collateral damage from modified ODNs may impede the proliferation of gene-edited human cells, posing challenges for therapeutic applications.