Murine inhalation model of cryptococcosis

For murine infection assays, 6-week-old female BALB/c mice (Animal Resources Centre, Australia) were infected by nasal inhalation [44]. For each strain (H99 and H99_CAS9), 10 mice were inoculated with a 50 μL drop containing 5 × 10⁵ C. neoformans cells. A maximum of 5 mice were housed per IVC cage (Tecniplast, USA) with bed-o’ cobs 1/8” bedding (The Andersons, USA), crink-L’ nest nesting material (The Andersons, USA), and cardboard as environmental enrichment. Mice were provided Rat and Mouse Cubes (Specialty Feeds, Australia) and water ad libitum. Each mouse was examined and weighed twice daily for the duration of the experiment, with affected mice euthanized via CO₂ inhalation once body weight had decreased to 80% of pre-infection weight or they exhibited symptoms consistent with infection. Death after CO₂ inhalation was confirmed by pedal reflex prior to dissection. The brain, lungs, liver, spleen and kidneys were collected, homogenized and plated to determine colony-forming units per gram organ weight. Kaplan-Meier survival curves were plotted using GraphPad Prism 7.0 (GraphPad Software, USA). Significance was analyzed using the log-rank test, while organ burden significance was determined using a one-way ANOVA with Tukey’s multiple comparisons test. P values of <0.05 were considered significant.