Abstract
Cryptococcus gattii is a primary fungal pathogen, which causes pulmonary and brain infections in healthy as well as immunocompromised individuals. Genetic manipulations in this pathogen are widely employed to study its biology and pathogenesis, and require integration of foreign DNA into the genome. Thus, identification of gene free regions where integrated foreign DNA can be expressed without influencing, or being influenced by, nearby genes would be extremely valuable. To achieve this goal, we examined publicly available genomes and transcriptomes of C. gattii, and identified two intergenic regions in the reference strain R265 as potential "safe haven" regions, named as CgSH1 and CgSH2. We found that insertion of a fluorescent reporter gene and a selection marker at these two intergenic regions did not affect the expression of their neighboring genes and were also expressed efficiently, as expected. Furthermore, DNA integration at CgSH1 or CgSH2 had no apparent effect on the growth of C. gattii, its response to various stresses, or phagocytosis by macrophages. Thus, the identified safe haven regions in C. gattii provide an effective tool for researchers to reduce variation and increase reproducibility in genetic experiments.