The development of a CRISPR-Cas9 gene editing system for Cryptococcus deneoformans
Du Plooy, Lukas Marthinus
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The pathogenic yeasts, Cryptococcus neoformans and C. deneoformans, are responsible for potentially fatal meningoencephalitis in immunocompromised individuals, most notably in patients who have AIDS. Only three drugs are commonly administered to treat cryptococcal infections and most are not readily available in developing countries most affected by the AIDS pandemic. Cheaper and more widely available drugs are therefore needed. Developing molecular methods to disable genes encoding virulence factors could help to elucidate the mechanism of action of potential drugs against these fungal pathogens. Previously, researchers mostly relied on biolistic transformation to deliver DNA into cells for homologous integration into the targeted site. Recent developments with CRISPR-Cas9-based systems for gene targeting made it possible to utilise another transformation technique, electroporation, to knock genes out. In this study, a one-step CRISPR- Cas9 system was developed to be delivered into cells with electroporation. This system consists out of two plasmids, carrying a nourseothricin and G418 resistance marker respectively, as well as a CAS9 gene. A third plasmid was used to construct guide DNA, which was then amplified and cloned into the two CRISPR-Cas9 plasmids carrying the CAS9 gene. The plasmids carrying the CRISPR-Cas9 components were maintained transiently for expression of the CRISPR-Cas9 genes before these constructs were degraded by the cells. Donor DNA was also constructed to remove parts of the biosynthetic genes ADE2 and HIS3 to obtain adenine and histidine auxotrophic mutants with visually discernible phenotypes. A second round of transformation can then introduce new donor DNA to repair these auxotrophic genes whilst disrupting virulence genes for virulence studies. Electroporation proved to be very inefficient in this study and gene targeting was unsuccessful. Using large amounts of plasmid and donor DNA yielded the best results, although no more than 8 colonies were seen on a few selective media agar plates. Inefficient transformation could be due to old and faulty electroporation equipment or ineffective delivery of the electrical current to cells. In the future, other transformation methods will be employed to deliver the plasmids constructed in this study into C. deneoformans cells. This system can then be used to remove virulence genes to study their role in infection, which could help to elucidate the mechanism of action behind potential drugs. For instance, a capsule-less mutant could reveal what effect a drug targeting capsule synthesis will have on the ability of these yeasts to cause disease.