Iterative marker excision system.

Area of Science:

• Genomics
• Molecular Biology
• Genetic Engineering

Background:

• Genomic engineering requires efficient deletion of large DNA fragments and gene knockouts.
• Removing antibiotic resistance markers post-engineering is crucial to avoid unintended effects.
• Existing methods may leave active recombinase sites, complicating further modifications.

Purpose of the Study:

• To establish a novel iterative marker excision system (IMES) for precise genomic engineering.
• To enable the removal of DNA fragments and antibiotic resistance markers without residual recombinase sites.
• To facilitate multi-step genomic modifications with high fidelity.

Main Methods:

• Development of the iterative marker excision system (IMES) based on phiC31 integrase and mutant att sites.
• Utilisation of inversely oriented B-CC and P-GG sites for DNA fragment deletion.
• Characterization of recombination efficiencies between engineered att sites (RR, B-CC, P-GG, LL).

Main Results:

• IMES demonstrated highly effective deletion of DNA fragments between B-CC and P-GG sites.
• The engineered RR site showed minimal recombination with itself and other att sites.
• Recombination efficiencies were significantly low (0.1% for RR-P-GG, 1% for RR-LL), preventing unwanted DNA rearrangements.
• Demonstrated fabrication of multi-purpose antibiotic cassettes and successful utilization of IMES.

Conclusions:

• IMES provides a robust solution for removing DNA fragments and selection markers in genomic engineering.
• The system allows for multistep engineering without the risk of undesired DNA recombination.
• IMES enhances the precision and efficiency of creating organisms with improved properties.