MYCOLab Ai Logo - gold and army green

Co-transformation of Aspergillus fumigatus: a simple and efficient strategy for gene editing without linking selectable markers

Summary

Scientists have developed a new technique for editing genes in a dangerous fungal pathogen called Aspergillus fumigatus. Instead of permanently attaching antibiotic resistance markers to the target genes (which can interfere with normal gene function), they use a clever strategy of introducing two different DNA pieces simultaneously. One piece makes the desired gene edit while the second introduces a resistance marker to a completely different location in the genome. This approach is simple, inexpensive, and works about 11% of the time, making it practical for identifying successfully edited strains.

Background

Aspergillus fumigatus is a globally prevalent human fungal pathogen causing invasive aspergillosis and other serious infections. Gene editing is critical for studying pathogen biology, but traditional methods linking selectable markers to target genes can disrupt flanking DNA sequences and alter gene expression patterns. This study addresses the need for marker-free gene editing strategies that maintain genomic context.

Objective

To develop and validate a co-transformation strategy in A. fumigatus that enables precise, marker-free gene edits at a locus of interest without disturbing flanking DNA sequences. The strategy uses simultaneous introduction of a marker-free modified gene construct and a plasmid directing selectable marker integration to a different genomic location.

Results

An average of 11% of pyrithiamine-resistant transformants were successfully co-transformed with the marker-free creA M construct, an efficiency comparable to CRISPR-based methods. Screening 10-20 selected transformants was sufficient for identifying the desired genetic modifications. PCR and restriction digest analysis confirmed successful integration of the modified creA construct in four independently isolated transformants.

Conclusion

Co-transformation represents a simple, inexpensive, and efficient method for generating unlinked marker-free genetic modifications in A. fumigatus without requiring additional reagents beyond standard transformation protocols. This strategy is particularly valuable for gene regulation studies and maintaining wild-type expression patterns, complementing current genetic modification toolsets.
  • Published in:Access Microbiology,
  • Study Type:Experimental Research,
  • Source: PMID: 41089555, DOI: 10.1099/acmi.0.001057.v3
Scroll to Top