Shahana Shahida, Childers Delma S, Ballou Elizabeth R, Bohovych Iryna, Odds Frank C, Gow Neil A R, Brown Alistair J P
School of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom.
PLoS One. 2014 Jun 18;9(6):e100390. doi: 10.1371/journal.pone.0100390. eCollection 2014.
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 CaMET3p-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.
精确的基因组修饰对于白色念珠菌的分子剖析至关重要，并且正在产生有关特定基因功能在这种人类主要真菌病原体中的作用的宝贵信息。白色念珠菌是天然二倍体，无法进行减数分裂，并使用非标准遗传密码。因此，必须开发专门的工具用于白色念珠菌中的基因破坏，以允许删除两个目标等位基因，并且在某些情况下，回收念珠菌特异性选择标记。此前，我们开发了一种基于Cre重组酶的工具，该工具通过loxP位点之间的位点特异性重组以90-100%的效率在白色念珠菌中回收标记。具有讽刺意味的是，该系统的实用性受到Cre极高效率的阻碍，这使得在大肠杆菌中构建携带两侧有loxP位点的甲硫氨酸可调节CaMET3p-cre基因的稳定破坏盒变得困难。因此，我们通过设计携带合成的、含内含子的cre基因的新Clox盒显著增强了该系统。Clox试剂盒促进了高效转化和标记回收，从而简化并加速了白色念珠菌中的基因破坏过程。事实上，纯合突变体可以在两周内产生并解决其标记问题。Clox试剂盒有助于涉及单标记回收或多标记基因破坏的策略。此外，它包括显性NAT1标记以及URA3、HIS1和ARG4盒，从而允许对临床分离株以及白色念珠菌的基因标记菌株进行操作。这种新的Clox系统提供的加速基因破坏策略可能会对剖析白色念珠菌致病生物学的速度产生深远影响。
