Abstract
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 ku80Δ 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.
Highlights
The discovery of the process of transformation was key to the development of the field of molecular genetics
For the Fractional Inhibitory Concentration (FIC) assays [31], inhibitors were serially diluted along the abscissa of a 96 well plate, while the DNA damaging agents were diluted along the ordinate
Z-5-(4-hydroxybenzylidene)-2-imino1,3-thiazolidin-4-one is a known inhibitor of Mre11, and 5,6-bis(((E)-benzylidene) amino)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (SCR7) targets DNA ligase IV [38,39,40,41]. In addition to these known non-homologous end joining (NHEJ) inhibitors, we chose to study a compound not directly associated with NHEJ—the poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor 1-(4((dimethylamino)methyl)phenyl)-8,9-dihydro-2,7,9a-triazabenzo[cd]azulen-6(7H)-one (AG14361), a tricyclic benzimidazole that inhibits single strand DNA repair
Summary
The discovery of the process of transformation was key to the development of the field of molecular genetics. Chemical Inhibitors Increase Targeted Construct Integration in Cryptococcus neoformans virulent strain, giving rise to the term transformation [1]. It was not until 1944 that Avery and colleagues used transformation to prove that this factor was DNA [2]. Beggs subsequently demonstrated that S. cerevisiae could maintain a plasmid carrying the 2μ origin of replication without the need for integration [4]. These discoveries established S. cerevisiae as the premier eukaryotic model for molecular genetics. Transformation protocols were subsequently developed for Neurospora crassa [5] and Aspergillus nidulans [6], and over the following decades, the development of transformation protocols made many previously intractable species easier to study
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