Efficient Homology-Directed Repair with Circular Single-Stranded DNA Donors

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR-based nucleases have advanced genome editing, but homology-directed repair (HDR) efficiency remains a challenge.
• Traditional DNA donors like plasmids have low HDR efficiency and off-target integration issues.
• Single-stranded DNA (ssDNA) donors show promise for efficient HDR with reduced off-target effects.

Purpose of the Study:

• To develop and evaluate circular ssDNA (cssDNA) donors produced via ssDNA phage for enhanced HDR efficiency.
• To compare the efficacy of cssDNA donors against linear ssDNA (lssDNA) donors with different CRISPR nucleases (Cas9, Cas12a).
• To assess precise gene editing and fluorescent tag knockin efficiencies in mammalian cell lines.

Main Methods:

• Production of long cssDNA donors using ssDNA phage technology.
• Development of a modified traffic light reporter (TLR-multi-Cas variant 1 [MCV1]) system for comparing nuclease and donor efficiencies.
• Evaluation of HDR efficiencies with cssDNA, lssDNA, and plasmid donors using Cas9 and Cas12a nucleases.
• Assessment of fluorescent tag knockin at endogenous loci in HEK293T and K562 cells.

Main Results:

• cssDNA donors demonstrated superior HDR integration frequencies compared to lssDNA donors.
• The TLR-MCV1 system enabled efficient side-by-side comparison of different nuclease and donor combinations.
• High targeting efficiency was achieved with cssDNA donors, enabling biallelic integrants.
• cssDNA donors facilitated robust and efficient insertion of reporter tags at endogenous sites.

Conclusions:

• cssDNA donors produced via ssDNA phage are highly efficient and cost-effective for homology-directed repair.
• Circular ssDNA donors represent an improved alternative to linear ssDNA and plasmid donors for gene knockins.
• This method offers a robust strategy for precise genome editing applications in mammalian cell lines.