Improving Precise Genome Editing Using Donor DNA/gRNA Hybrid Duplex Generated by Complementary Bases.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR/Cas9 enables precise genome editing via DNA double-strand breaks (DSBs).
• Homology-directed repair (HDR) uses donor DNA for precise editing but is often inefficient.
• Low HDR efficiency limits the widespread application of CRISPR-based genome editing.

Purpose of the Study:

• To develop a novel donor DNA/guide RNA (gRNA) hybrid duplex (DGybrid) to enhance HDR efficiency.
• To improve the concentration of donor DNA at the DNA cleavage site.
• To create a universal and concise tool for efficient genome editing.

Main Methods:

• Constructed a DGybrid by combining a sequence-extended gRNA with a single-stranded oligodeoxynucleotide (ssODN).
• Evaluated DGybrid efficiency in *Saccharomyces cerevisiae* for HDR-mediated genome editing.
• Assessed nucleic acid introduction efficiency using flow cytometry.

Main Results:

• Achieved a 1.8-fold improvement in HDR-mediated editing efficiency (from 35% to 62%) compared to separate introduction of gRNA and donor DNA.
• Demonstrated efficient co-incorporation of RNA and ssODNs into cells via the DNA/RNA hybrid.
• Confirmed the effectiveness of the DGybrid in enhancing genome editing outcomes.

Conclusions:

• The DGybrid technique significantly boosts HDR efficiency in CRISPR/Cas9 genome editing.
• This method facilitates simultaneous delivery and cellular uptake of essential editing components.
• DGybrid presents a promising, universally applicable tool for advancing precise genome engineering.