Efficient and Precise Integration of Large DNA Sequences Using Precise Interstrand Cross-Linking of Long ssDNA and...

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Homology-directed repair (HDR) is crucial for precise genome editing but faces limitations in large DNA sequence editing, including low efficiency and off-target effects.
• Current methods for preparing DNA donors for HDR, such as phosphorylation, have inherent stability and efficiency issues.

Purpose of the Study:

• To develop a novel strategy to enhance the efficiency and accuracy of homology-directed repair (HDR) for large DNA sequence gene editing.
• To overcome the limitations of low efficiency and off-target effects associated with current HDR methods.

Main Methods:

• Synthesized chemically modified long single-stranded DNA (lssDNA) using a novel AOLP method for enhanced stability.
• Developed a light-activated ligation strategy using cyanovinylcarbazole nucleoside (^CNVK) for precise interstrand cross-linking between lssDNA and sgRNA.
• Utilized Cas9-based gene editing to induce double-strand breaks (DSBs) and facilitate HDR.

Main Results:

• The novel lssDNA donors demonstrated 4- to 12-fold higher knock-in (KI) efficiency compared to conventional phosphorylated donors in K562, HEK293T, and HepG2 cells.
• Achieved a >4.7-fold enhancement in HDR KI accuracy in HEK293T cells compared to previous commercial lssDNA methods.
• Successfully inserted a gene-sized 1.4 kilobase lssDNA with an unprecedented KI rate of approximately 36% in HEK293T cells.

Conclusions:

• The developed light-activated ligation strategy with chemically modified lssDNA significantly enhances HDR efficiency and accuracy for large DNA insertions.
• This approach offers a promising solution for overcoming existing challenges in genome editing, enabling precise and efficient large-scale genetic modifications.