An intergenic "safe haven" region in Aspergillus fumigatus.

Aspergillus fumigatus is the most common opportunistic human fungal pathogen responsible for invasive aspergillosis. Gene manipulation is critical for the investigation of A. fumigatus biology and pathogenesis at the molecular level, and it often requires integration of the introduced DNA into the fungal genome. Here we have searched and identified two potential "safe haven" regions, SH1 and SH2, based on A. fumigatus genome annotation and transcriptome data. When a DNA fragment carrying a fluorescent protein gene mNeonGreen (mNG) and a drug selection marker was inserted into SH1 or SH2, the expression of mNeonGreen was easily detected, indicating that SH1 and SH2 are not surpressive genetic regions. We found that insertion of this DNA fragment into SH1 did not cause any significant changes in the expression of neighboring genes. Insertion of this DNA into either SH1 or SH2 did not significantly alter any of the phenotypes that we analyzed comparing to the wild type control. By comparison, transformants with random ectopic integration of the same DNA fragment showed a wider range of variation in mNeonGreen expression and in virulence in an insect infection model. Having identified predetermined "safe-haven" regions in A. fumigatus could therefore help reduce experimental variations and increase reproducibility, as it has been for the C. neoformans field.