Cellular responses to targeted genomic sequence modification using single-stranded oligonucleotides and zinc-finger...

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• Sequence-specific genome modification is crucial for genetic research and therapies.
• Two primary methods, single-stranded oligonucleotides (ssODNs) and zinc-finger nucleases (ZFNs), are employed for precise gene editing.
• The cellular response to ssODN-mediated gene correction remains less understood compared to ZFNs.

Purpose of the Study:

• To investigate and compare cellular responses following ssODN and ZFN mediated genome modification.
• To determine the impact of each gene editing strategy on cell cycle progression and DNA damage.
• To elucidate the mechanisms underlying ssODN-mediated gene correction and its associated cellular consequences.

Main Methods:

• Human cells were utilized to compare ssODN and ZFN mediated correction of a genomic reporter gene.
• Cell cycle distribution analysis was performed on corrected cells.
• Phosphorylation of H2AX (a DNA damage marker) and direct detection of genomic double-strand breaks (DSBs) were quantified.
• The role of the mismatch repair (MMR) system in ssODN mediated correction was assessed.

Main Results:

• ssODN corrected cells exhibited cell cycle arrest in late S and G2/M phases, unlike ZFN corrected cells with normal profiles.
• Increased H2AX phosphorylation and unrepaired genomic DSBs were observed in ssODN corrected cells.
• ZFN corrected cells showed significantly lower rates of DNA damage and normal cell cycle progression.
• While MMR deficiency enhanced ssODN correction, it did not affect the proliferation of corrected cells.

Conclusions:

• ssODN mediated gene correction activates DNA damage signaling and causes cell cycle arrest due to unrepaired DSBs.
• ZFNs provide a safer genome modification strategy with normal cell cycle distribution and minimal DNA damage.
• Understanding these distinct cellular responses is critical for selecting appropriate gene editing tools for therapeutic applications.