Expanding the DNA editing toolbox: Novel lambda integrase variants targeting microalgal and human genome sequences

Area of Science:

• Synthetic biology
• Molecular biology
• Biotechnology

Background:

• Recombinase enzymes efficiently integrate large DNA, but their specificity limits applications.
• Engineering is needed to overcome sequence limitations and enhance robustness.

Purpose of the Study:

• To evolve novel lambda integrase variants for targeting non-natural DNA sequences.
• To enable precise genomic integration in key microorganisms for biomanufacturing and carbon sequestration.

Main Methods:

• Directed evolution of lambda integrase.
• In vitro specificity assays.
• In vitro and in human cell assays for targeting engineered sites.

Main Results:

• Developed lambda integrase variants with >1000-fold specificity switch for non-natural target sites.
• Demonstrated efficient targeting of a human genomic site homologous to an algal sequence.
• Showcased successful in vitro and in human cell targeting with engineered integrases.

Conclusions:

• Engineered integrase variants offer expanded DNA editing capabilities.
• These variants are valuable for targeted genomic insertion of large DNA cargos.
• Potential applications include green biomanufacturing and carbon sequestration using engineered microorganisms.