Screening and characterization of DNA aptamers that modulate prime editing

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• CRISPR-Cas9 is a key tool for gene therapy but suffers from low homologous recombination rates.
• Advanced gene-editing technologies like prime editors (PE) aim to overcome CRISPR limitations.
• Cas9-specific single-stranded DNA (ssDNA) aptamers were investigated for their potential to improve PE2 functionality.

Purpose of the Study:

• To investigate if Cas9-specific ssDNA aptamers can enhance the efficiency of the PE2 prime editing system.
• To evaluate the therapeutic potential of aptamer-enhanced PE2 in restoring gene function in cancer cells.

Main Methods:

• Systematic evolution of ligands by exponential enrichment (SELEX) was used to isolate Cas9-specific ssDNA aptamers.
• Molecular docking simulations characterized aptamer-PE2 interactions.
• PE2 editing efficiency was assessed using flow cytometry and Sanger sequencing; p53 repair was evaluated via qPCR and Western blot; cellular responses were measured by CCK-8 and apoptosis assays.

Main Results:

• Molecular docking identified specific interaction sites between aptamers and PE2.
• Incorporation of aptamers significantly boosted PE2 editing efficiency.
• The aptamer-PE2 complex successfully restored p53 function in bladder cancer cells, reducing proliferation and increasing apoptosis.

Conclusions:

• Cas9-specific aptamers effectively enhance prime editing efficiency.
• These findings offer new strategies for modulating prime editing and improving its clinical applicability in gene therapy.