Enhancing the specificity of recombinase-mediated genome engineering through dimer interface redesign.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Site-specific endonucleases have advanced genome engineering.
• Hybrid recombinases offer potential for targeted genetic payload delivery.
• Current hybrid recombinases suffer from low specificity due to homodimer formation.

Purpose of the Study:

• To engineer enhanced hybrid recombinases with improved targeting specificity.
• To reduce off-target modifications caused by undesirable homodimers.
• To assess the utility of enhanced recombinases for gene delivery in mammalian cells.

Main Methods:

• Rational design and directed evolution of serine recombinase dimerization interfaces.
• Generation of a novel recombinase architecture to minimize homodimer formation.
• Testing enhanced recombinases in mammalian cells for specificity and integration rates.

Main Results:

• Engineered recombinases showed >500-fold reduction in undesirable homodimer formation.
• Enhanced recombinases demonstrated substantially improved targeting specificity in mammalian cells.
• Site-specific integration rates were comparable to site-specific nucleases.
• Low toxicity and high specificity were observed for delivering coagulation factor IX and α-galactosidase genes.

Conclusions:

• Protein engineering can significantly enhance hybrid recombinase specificity.
• Enhanced recombinases offer a promising tool for precise genome engineering.
• These enzymes hold potential for both basic research and therapeutic applications in gene delivery.