Applying CRISPR/Cas9 to identify virulence factors in the pathogenic fungus, Cryptococcus gattii
Cryptococcus gattii is a startling example of the unpredictability of fungal pathogens. Dogma suggested this pathogen was restricted to tropical and subtropical regions of the world, infecting mostly immunocompromised mammals. However, starting in 1999 and continuing still today, C. gattii was discovered as the causative agent of an outbreak in the Pacific Northwest (PNW), with cases being reported from Vancouver Island, British Columbia, then in mainland Canada, and eventually in Washington state and Oregon. This novel emergence demonstrated C. gattii’s ability to adapt and thrive in a temperate climate as well as manifest with unfamiliar clinical presentations. Clinical infections presented predominantly as a pulmonary infection, even in immunocompetent hosts as opposed to the typical fungal meningitis infection observed in the tropics in immunocompromised mammals. Here we applied CRISPR/Cas9 genome editing technology in an effort to disrupt a putative collagen binding domain, cDNA-26, suspected of conveying an increased binding ability of a collagenase, subsequently increasing virulence in one of the three genotypes observed in the PNW emergence. Yeast cells were cultured from a 2006 clinical PNW infection and electroporated in an effort to transform these cells with transient linear DNA cassettes containing a yeast-optimized Cas9 encoding gene and a gRNA sequence unique to the cDNA-26 exon sequence. Post-transformation DNA was extracted and results were assessed using real time PCR (rtPCR). After receiving inconclusive results, amplicon sequencing was utilized to further inspect the DNA for gene disruption. The amplicon sequencing analysis revealed our initial attempt at guided genome editing had failed. Further troubleshooting this procedure will include the verification of our in-house electroporation protocol, sequencing of our DNA cassette in order to ensure correct design, and rtPCR from an RNA extraction to ensure the translation and production of our Cas9 protein. Once transformation is confirmed, an RNA analysis spanning intronic regions will be used to assess the extent of our knockout. Future research will include a mouse model study to compare the virulence of our transformed strain with the wild type to determine if the cDNA-26 domain under question is indeed conveying the increased virulence.