Efficiency of genome editing using modified single-stranded oligodeoxyribonucleotides in human cells
Seryoung Kim1, Yosuke Matsushita2,3, Toyomasa Katagiri2,3
1Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, 1-8- 31 Midorigaoka, Ikeda, 563-8577, Osaka, Japan.
Scientific Reports
|March 22, 2025
Summary
Optimizing single-stranded oligodeoxyribonucleotide (ssODN) gene editing with locked nucleic acid (LNA) modifications significantly enhances efficiency. This study demonstrates LNA integration in ssODNs boosts precise gene editing outcomes for therapeutic applications.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Single-stranded oligodeoxyribonucleotide (ssODN) gene editing shows therapeutic promise but requires efficiency improvements.
- Optimizing ssODN design is crucial for effective genome editing applications.
Purpose of the Study:
- To investigate the impact of strand length and locked nucleic acid (LNA) modifications on ssODN gene editing efficiency.
- To identify optimal LNA positions and lengths for enhancing precise base deletions and substitutions.
Main Methods:
- Utilized an assay cassette in HEK293T cells to measure precise eight-base deletions via ssODN editing.
- Systematically introduced LNA modifications at various positions within 70 nt and 90 nt ssODNs.
- Evaluated editing efficiency for both deletions and base substitutions.
Main Results:
- ssODNs with specific LNA modifications (e.g., five pairs at 25-35 nt) demonstrated an approximately 18-fold increase in editing efficiency compared to unmodified ssODNs.
- A 90 nt ssODN with optimized LNA placement achieved the highest editing efficiency.
- LNA modifications were also effective for base substitutions, achieving 0.63% efficiency per cell.
Conclusions:
- Locked nucleic acid modifications represent a powerful strategy for significantly enhancing ssODN-mediated genome editing efficiency.
- Optimized LNA placement and ssODN length are critical factors for maximizing precise gene editing outcomes.
- This approach holds potential for advancing therapeutic gene editing strategies through improved oligo design.
