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

Authors

Shahida Shahana, University of Aberdeen
Delma S. Childers, University of Aberdeen
Elizabeth R. Ballou, University of Aberdeen
Iryna Bohovych, University of Nebraska- LincolnFollow
Frank C. Odds, University of Aberdeen
Neil A.R. Gow, University of Aberdeen
Alistair J.P. Brown, University of AberdeenFollow

Document Type

Article

Date of this Version

2014

Citation

Shahana S, Childers DS, Ballou ER, Bohovych I, Odds FC, et al. (2014) New Clox Systems for Rapid and Efficient Gene Disruption in Candida albicans. PLoS ONE 9(6): e100390.

Comments

Copyright 2014 Shahana et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License

Abstract

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.