Rampant transposition following RNAi loss cause hypermutation and antifungal drug resistance in Cryptococcus neoformans

In this study, two clinical hypermutator C. neoformans isolates, Bt65 and Bt81, were identified. These two strains displayed significantly increased mutation rates on antifungal drug media. The majority of the de novo drug resistance in these two strains is mediated by insertions of the Cnl1 transposon into genes whose mutation confers drug resistance. Genetic backcrossing, quantitative trait loci mapping, and CRISPR-mediated genetic complementation all confirmed that a nonsense mutation in the RNAi component ZNF3, resulting in loss of RNAi is the cause of the increased transposition in these two hypermutator strains. Whole-genome sequencing revealed both Bt65 and Bt81 encode >800 copies or fragments of the Cnl1 element. Sequencing of small RNAs also confirmed the role of Znf3 in the silencing of Cn1l. Together, these results demonstrate that the hypermutator phenotype in Bt65 and Bt81 is attributable to loss of RNAi, allowing rampant transposition of Cnl1, which can result in antifungal drug resistance.