An auxin-inducible degron system for conditional mutation in the fungal meningitis pathogen Cryptococcus neoformans

Cryptococcus neoformans is the top-ranked W.H.O. fungal priority pathogen, but tools for generating conditional mutations are limited. Auxin-inducible degron systems permit rapid and effective cellular depletion of a tagged protein of interest upon adding a small molecule. These tools are invaluable, particularly for studying essential genes, which may play important roles in pathogen biology. AID2 is one such system that improves on previous strategies. This system achieves greater sensitivity and specificity using a "bumped" auxin, 5-Ph-IAA, alongside an OsTIR1(F74G) "hole" mutant. We adapted the AID2 system for C. neoformans by codon optimizing OsTIR1(F74G) and tested its use in multiple scenarios. We demonstrate that the C. neoformans optimized AID2 system enables effective degradation of proteins, including essential proteins, and can be used to discriminate essential from non-essential genes. This tool enables the study of unexplored parts of the C. neoformans genome.