Study of HST6 function in Candida albicans through the application of a CRISPR-Cas9 gene editing system
The aim of this project was to construct a HST6 double deletion mutant of Candida albicans to elucidate the role and function of this gene. HST6 is an ABC transporter and homologue of Ste6 (Saccharomyces cerevisiae) which plays a role in mating in this organism. The first method used was the SAT1 flipper system, which alters genes using an antibiotic resistant marker (Nourseothricin) to select for integration. Homozygous mutants are created by transforming the organism with DNA containing the FLP gene which are flanked by target sequences found in the genome of C. albicans. Two rounds of integration are required as this organism has a duplicate set of genes and both copies needs to be removed sequentially. For the second round of integration, the mutant is grown on Maltose to flip out and regenerate the nourseothricin resistant marker. In this study a previously constructed plasmid was used to delete the HST6 gene, but after various attempts, no success was achieved. Transformations were unsuccessful and after molecular confirmation protocols such as PCR was performed, the gene remained intact. While using the SAT1 flipper system, the article by (Vyas et al., 2015) was published and the CRISPR-Cas 9 method was used for gene deletion in C. albicans. This system relies upon the use of the endonuclease (Cas9) and guide RNA to create double stranded cleaving. This system wasn’t available to order, and so a similar system was constructed. While in the process to develop this system, another article was published, describing a CRISPR-Cas gene editing system. This system was however made available to the wider scientific community through depositing of all plasmids at Addgene (www.addgene.org). This system was subsequently obtained via Addgene and a proof of concept, the ADE2 gene was targeted as the double deletion mutant showed an easily observed phenotype where if both alleles are deleted, colonies turn from white to red. After several attempts, a double deletion mutant of ADE2 was constructed in C. albicans using the CRISPR-Cas9 system and red colonies were observed. This allowed for optimisation of the system which led to the HST6 gene to be targeted. After confirmation via PCR, the HST6 gene was removed and it was managed to create a complemented strain where the HST6 gene was added back into the genome of the organism. The wild-type strain, double deletion mutant and complemented strain was used to determine the influence of fluconazole on the different variants of the organism. The XTT assay revealed that the biofilm metabolic activity of the wild type strain was less influenced by fluconazole (55.8% reduction) as the biofilm metabolic activity of the mutants (75.1 % and 70.3 % respectively). It can be assumed that HST6 plays a role in fluconazole resistance, but further investigation is required as no significant difference was obtained between reduction in biofilm metabolic activity because of fluconazole addition, between the double deletion mutant and the complemented strain. Nonetheless, a CRISPR-Cas9 gene editing system was used effectively to construct both a double deletion mutant of HST6 as well as a complemented strain in the diploid C. albicans. This system will allow for more genes to be targeted in the future and might lead to a solution to drug resistance in C. albicans.