Targeting specific DNA G-quadruplexes with CRISPR-guided G-quadruplex-binding proteins and ligands

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• DNA G-quadruplexes (G4s) are crucial in biological processes and diseases.
• Existing technologies lack precision in manipulating specific G4 structures for functional studies and therapeutic development.

Purpose of the Study:

• To develop a novel technology for targeted manipulation of specific DNA G-quadruplex folding.
• To investigate the biological functions of specific G4s using precise manipulation techniques.
• To explore potential therapeutic applications by controlling G4 structures.

Main Methods:

• Utilized CRISPR technology combined with G4-stabilizing proteins (e.g., nucleolin) and ligands.
• Engineered catalytically inactive Cas9 fused with nucleolin to target and stabilize specific G4s in genomic loci.
• Employed CRISPR to enhance the selectivity of G4-binding compounds (pyridodicarboxamide, pyridostatin) for intra-G4 targeting.

Main Results:

• Successfully stabilized G4s in the MYC oncogene promoter, Itga7 gene promoter, and telomeres using CRISPR-nucleolin fusion.
• Demonstrated that targeted G4 stabilization leads to significant cellular effects: proliferation arrest, inhibited myoblast differentiation, and cell senescence.
• Achieved enhanced selectivity of G4 ligands, enabling more precise investigation of de novo G4 functions compared to traditional methods.

Conclusions:

• The developed CRISPR-based system provides unprecedented precision in manipulating DNA G-quadruplex folding at specific genomic sites.
• This technology offers a powerful tool for dissecting the functional roles of G4s in various biological contexts.
• The findings pave the way for advancing G4-related research and developing targeted therapeutic strategies for diseases involving G4s.