Chemical Inhibitors of Non-Homologous End Joining Increase Targeted Construct Integration in Cryptococcus neoformans

被引:25
作者
Arras, Samantha D. M. [1 ]
Fraser, James A. [1 ]
机构
[1] Univ Queensland, Sch Chem & Mol Biosci, Australian Infect Dis Res Ctr, St Lucia, Qld 4072, Australia
来源
PLOS ONE | 2016年 / 11卷 / 09期
基金
英国医学研究理事会;
关键词
DEPENDENT PROTEIN-KINASE; STRAND BREAK REPAIR; DNA-PK; HOMOLOGOUS RECOMBINATION; ASPERGILLUS-FUMIGATUS; INFECTIOUS-DISEASES; PNEUMOCOCCAL TYPES; REPLICATION FORKS; HYBRID PLASMID; GENE DELETION;
D O I
10.1371/journal.pone.0163049
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
The development of a biolistic transformation protocol for Cryptococcus neoformans over 25 years ago ushered in a new era of molecular characterization of virulence in this previously intractable fungal pathogen. However, due to the low rate of homologous recombination in this species, the process of creating targeted gene deletions using biolistic transformation remains inefficient. To overcome the corresponding difficulty achieving molecular genetic modifications, members of the Cryptococcus community have investigated the use of specific genetic backgrounds or construct design strategies aimed at reducing ectopic construct integration via non-homologous end joining (NHEJ). One such approach involves deletion of components of the NHEJ-associated Ku heterodimer. While this strategy increases homologous recombination to nearly 100%, it also restricts strain generation to a ku80 Delta genetic background and requires subsequent complex mating procedures to reestablish wild-type DNA repair. In this study, we have investigated the ability of known inhibitors of mammalian NHEJ to transiently phenocopy the C. neoformans Ku deletion strains. Testing of eight candidate inhibitors revealed a range of efficacies in C. neoformans, with the most promising compound (W7) routinely increasing the rate of gene deletion to over 50%. We have successfully employed multiple inhibitors to reproducibly enhance the deletion rate at multiple loci, demonstrating a new, easily applied methodology to expedite acquisition of precise genetic alterations in C. neoformans. Based on this success, we anticipate that the use of these inhibitors will not only become widespread in the Cryptococcus community, but may also find use in other fungal species as well.
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页数:14
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