Gene disruption in Candida albicans using a synthetic, codon-optimised Cre-loxP system

The development of the molecular toolbox for the fungal pathogen Candida albicans has been hampered by its lack of an exploitable sexual cycle, its diploid nature, and its non-canonical genetic code. We describe the adaptation of the Cre-loxP site-specific recombination system as a tool for the efficient and controlled disruption of C. albicans genes. We have validated this system by disrupting two C. albicans loci: ADE2 and MET15. Ade2 and met15 null mutants were made using loxP-flanked ARG4- and HIS1-based disruption cassettes. These markers were then resolved from the C. albicans genome using a synthetic codon-optimised cre recombinase gene, with near 100% efficiency. Finally, CIp plasmids containing the URA3, HIS1, and ARG4 markers were generated for the reintegration of markers and target genes in control strains. This system allows multiple and sequential genetic manipulations, which will facilitate the functional analysis of multigene families in C. albicans.