Homology-directed repair (HDR) is a critical pathway for achieving precise genetic modifications in CRISPR-based genome editing. Synthetic single-stranded DNA (ssDNA) donors have become the preferred choice for guiding HDR, enabling knock-ins, point mutations, and small insertions with high accuracy.

Advantages of ssDNA Donors

Compared with double-stranded DNA (dsDNA), ssDNA offers:

● Higher knock-in efficiency: Single-stranded templates are more readily incorporated by HDR machinery.

● Reduced off-target integration: Lower risk of random insertions minimizes experimental variability.

● Flexibility in design: Supports point mutations, short tags, and small deletions.

Research Applications

● Gene therapy models: Correcting pathogenic variants in patient-derived cells.

● Reporter gene knock-ins: Tagging endogenous loci with fluorescent or epitope markers for functional studies.

● Functional genomics: Creating precise mutations to study gene function or regulatory elements.

Technical Considerations

Long ssDNA can be prone to degradation, and efficient cellular delivery remains challenging. Optimized design, chemical modifications (e.g., phosphorothioate linkages), and high-quality synthesis are crucial for successful HDR experiments.

GenCefe Biotech Solutions

GenCefe Biotech provides custom ssDNA synthesis with high purity and flexible lengths, tailored for HDR and knock-in applications. Our solutions help researchers achieve precise and reproducible genome modifications, accelerating functional studies and therapeutic research.