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Precise genome modification is essential for the molecular dissection of  Candida albicans , and is yielding invaluable information about the roles of specific gene functions in this major fungal pathogen of humans.  C. albicans  is naturally diploid, unable to undergo meiosis, and utilizes a non-canonical genetic code. Hence, specialized tools have had to be developed for gene disruption in  C. albicans  that permit the deletion of both target alleles, and in some cases, the recycling of the  Candida- specific selectable markers. Previously, we developed a tool based on the Cre recombinase, which recycles markers in  C. albicans  with 90–100% efficiency via site-specific recombination between  loxP  sites. Ironically, the utility of this system was hampered by the extreme efficiency of Cre, which prevented the construction in  Escherichia coli  of stable disruption cassettes carrying a methionine-regulatable  CaMET3 p -cre  gene flanked by  loxP  sites. Therefore, we have significantly enhanced this system by engineering new  Clox  cassettes that carry a synthetic, intron-containing  cre  gene. The  Clox  kit facilitates efficient transformation and marker recycling, thereby simplifying and accelerating the process of gene disruption in  C. albicans . Indeed, homozygous mutants can be generated and their markers resolved within two weeks. The  Clox  kit facilitates strategies involving single marker recycling or multi-marker gene disruption. Furthermore, it includes the dominant  NAT1  marker, as well as  URA3, HIS1  and  ARG4  cassettes, thereby permitting the manipulation of clinical isolates as well as genetically marked strains of  C. albicans . The accelerated gene disruption strategies afforded by this new  Clox  system are likely to have a profound impact on the speed with which  C .  albicans  pathobiology can be dissected.

New Clox Systems for Rapid and Efficient Gene Disruption in Candida albicans