Danzeisen, E. L.; Lihon, M. V.; Milholland, K. L.; Bias, T. R.; Bates, A. F.; Hall, M. C.

The auxin-inducible degron (AID) technology is a convenient and powerful tool for protein functional characterization in a broad array of eukaryotic species. We recently demonstrated that the original AID and improved AID2 systems are very effective at rapid protein depletion in Candida albicans and described a limited set of reagents for their use in certain auxotrophic lab strains. With an eye towards broader applicability with improved flexibility, we report here a new series of template vectors suitable for employing AID2 technology in prototrophic C. albicans strains, including clinical isolates. We adapted a common recyclable antibiotic marker system for the required genome editing steps and developed a strategy for simultaneous CRISPR/Cas9-mediated tagging of both target alleles. We also developed a composite all-in-one tagging cassette that combines the degron tag and the OsTIR1F74A gene for single step strain engineering. We added a fluorescent protein tag option and designed and validated an approach for N-terminal tagging that retains natural promoter control. We also compared effectiveness of the two commonly used synthetic auxins, 5-Ph-IAA and 5-Ad-IAA and the two common OsTIR1 variants, F74A and F74G, and provide guidelines for using the new AID2 system. Finally, using the novel all-in-one cassette, we demonstrate that the AID2 system also works in Candida auris. The new reagents should enhance the convenience and accessibility of the AID2 system for the Candida research community.