{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,31]],"date-time":"2026-03-31T18:40:45Z","timestamp":1774982445580,"version":"3.50.1"},"reference-count":52,"publisher":"Oxford University Press (OUP)","issue":"6","license":[{"start":{"date-parts":[[2025,4,7]],"date-time":"2025-04-07T00:00:00Z","timestamp":1743984000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2025,6,4]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:p>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 through an auxin derivative, 5-Ph-IAA, alongside an OsTIR1F74G mutant. We adapted the AID2 system for C. neoformans by codon optimizing OsTIR1F74G 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 help discriminate essential from nonessential genes. This tool enables the study of unexplored parts of the C. neoformans genome.<\/jats:p>","DOI":"10.1093\/g3journal\/jkaf071","type":"journal-article","created":{"date-parts":[[2025,4,8]],"date-time":"2025-04-08T02:42:19Z","timestamp":1744080139000},"source":"Crossref","is-referenced-by-count":8,"title":["An auxin-inducible degron system for conditional mutation in the fungal meningitis pathogen <i>Cryptococcus neoformans<\/i>"],"prefix":"10.1093","volume":"15","author":[{"given":"Manning Y","family":"Huang","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Biophysics, University of California San Francisco , San Francisco, CA 94158 ,","place":["USA"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9462-3297","authenticated-orcid":false,"given":"Matthew J","family":"Nalley","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biophysics, University of California San Francisco , San Francisco, CA 94158 ,","place":["USA"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Patrick C","family":"Hecht","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biophysics, University of California San Francisco , San Francisco, CA 94158 ,","place":["USA"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7400-6657","authenticated-orcid":false,"given":"Hiten D","family":"Madhani","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biophysics, University of California San Francisco , San Francisco, CA 94158 ,","place":["USA"]}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"286","published-online":{"date-parts":[[2025,4,7]]},"reference":[{"issue":"15","key":"2025060406574381400_jkaf071-B1","doi-asserted-by":"publisher","first-page":"8927","DOI":"10.1073\/pnas.95.15.8927","article-title":"Systematic identification of essential genes by in vitro mariner mutagenesis","volume":"95","author":"Akerley","year":"1998","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"6","key":"2025060406574381400_jkaf071-B2","doi-asserted-by":"publisher","first-page":"e10207","DOI":"10.15252\/msb.202110207","article-title":"A genome-scale yeast library with inducible expression of individual genes","volume":"17","author":"Arita","year":"2021","journal-title":"Mol Syst Biol"},{"issue":"4","key":"2025060406574381400_jkaf071-B3","doi-asserted-by":"publisher","first-page":"e0122916","DOI":"10.1371\/journal.pone.0122916","article-title":"A genomic safe haven for mutant complementation in Cryptococcus neoformans","volume":"10","author":"Arras","year":"2015","journal-title":"PLoS One"},{"issue":"3","key":"2025060406574381400_jkaf071-B4","doi-asserted-by":"publisher","first-page":"e0016322","DOI":"10.1128\/msphere.00163-22","article-title":"FKS1 is required for Cryptococcus neoformans fitness in vivo: application of copper-regulated gene expression to mouse models of Cryptococcosis","volume":"7","author":"Beattie","year":"2022","journal-title":"mSphere"},{"key":"2025060406574381400_jkaf071-B5","doi-asserted-by":"publisher","author":"Billmyre","year":"2024","DOI":"10.1101\/2024.07.28.605507"},{"key":"2025060406574381400_jkaf071-B6","doi-asserted-by":"publisher","author":"Boucher","year":"2024","DOI":"10.1101\/2024.10.22.619677"},{"issue":"7","key":"2025060406574381400_jkaf071-B7","doi-asserted-by":"publisher","first-page":"774","DOI":"10.1021\/acssynbio.5b00284","article-title":"FlowCal: a user-friendly, open source software tool for automatically converting flow cytometry data from arbitrary to calibrated units","volume":"5","author":"Castillo-Hair","year":"2016","journal-title":"ACS Synth Biol"},{"issue":"5","key":"2025060406574381400_jkaf071-B8","doi-asserted-by":"publisher","first-page":"910","DOI":"10.1016\/j.cell.2018.01.035","article-title":"Pervasive, coordinated protein-level changes driven by transcript isoform switching during meiosis","volume":"172","author":"Cheng","year":"2018","journal-title":"Cell"},{"key":"2025060406574381400_jkaf071-B9","doi-asserted-by":"publisher","first-page":"103241","DOI":"10.1016\/j.fgb.2019.103241","article-title":"Amds as a dominant recyclable marker in Cryptococcus neoformans","volume":"131","author":"Erpf","year":"2019","journal-title":"Fungal Genet Biol"},{"issue":"4","key":"2025060406574381400_jkaf071-B10","doi-asserted-by":"publisher","first-page":"1357","DOI":"10.1534\/genetics.117.300656","article-title":"Multiple applications of a transient CRISPR-Cas9 coupled with electroporation (TRACE) system in the Cryptococcus neoformans species complex","volume":"208","author":"Fan","year":"2018","journal-title":"Genetics"},{"issue":"11","key":"2025060406574381400_jkaf071-B11","doi-asserted-by":"publisher","first-page":"1056","DOI":"10.1016\/j.fgb.2011.07.005","article-title":"Application of the systematic \u201cDAmP\u201d approach to create a partially defective C. albicans mutant","volume":"48","author":"Finkel","year":"2011","journal-title":"Fungal Genet Biol"},{"issue":"4","key":"2025060406574381400_jkaf071-B12","doi-asserted-by":"publisher","first-page":"211","DOI":"10.1038\/s41579-023-00861-x","article-title":"The WHO fungal priority pathogens list as a game-changer","volume":"21","author":"Fisher","year":"2023","journal-title":"Nat Rev Microbiol"},{"key":"2025060406574381400_jkaf071-B13","doi-asserted-by":"publisher","author":"Fu","year":"2024","DOI":"10.1101\/2024.11.29.626100"},{"issue":"6896","key":"2025060406574381400_jkaf071-B14","doi-asserted-by":"publisher","first-page":"387","DOI":"10.1038\/nature00935","article-title":"Functional profiling of the Saccharomyces cerevisiae genome","volume":"418","author":"Giaever","year":"2002","journal-title":"Nature"},{"issue":"1","key":"2025060406574381400_jkaf071-B15","doi-asserted-by":"publisher","first-page":"31","DOI":"10.1038\/nprot.2007.13","article-title":"High-efficiency yeast transformation using the LiAc\/SS carrier DNA\/PEG method","volume":"2","author":"Gietz","year":"2007","journal-title":"Nat Protoc"},{"issue":"8","key":"2025060406574381400_jkaf071-B16","doi-asserted-by":"publisher","first-page":"531","DOI":"10.1016\/j.fgb.2006.02.007","article-title":"Improvements to gene deletion in the fungal pathogen Cryptococcus neoformans: absence of Ku proteins increases homologous recombination, and co-transformation of independent DNA molecules allows rapid complementation of deletion phenotypes","volume":"43","author":"Goins","year":"2006","journal-title":"Fungal Genet Biol"},{"issue":"7955","key":"2025060406574381400_jkaf071-B17","doi-asserted-by":"publisher","first-page":"190","DOI":"10.1038\/s41586-023-05856-5","article-title":"Structural and mechanistic insights into fungal \u03b2-1,3-glucan synthase FKS1","volume":"616","author":"Hu","year":"2023","journal-title":"Nature"},{"issue":"1","key":"2025060406574381400_jkaf071-B18","doi-asserted-by":"publisher","first-page":"iyab180","DOI":"10.1093\/genetics\/iyab180","article-title":"Short homology-directed repair using optimized Cas9 in the pathogen Cryptococcus neoformans enables rapid gene deletion and tagging","volume":"220","author":"Huang","year":"2022","journal-title":"Genetics"},{"issue":"2","key":"2025060406574381400_jkaf071-B19","doi-asserted-by":"publisher","first-page":"e02334-14","DOI":"10.1128\/mBio.02334-14","article-title":"Essential gene discovery in the basidiomycete Cryptococcus neoformans for antifungal drug target prioritization","volume":"6","author":"Ianiri","year":"2015","journal-title":"mBio"},{"issue":"24","key":"2025060406574381400_jkaf071-B20","doi-asserted-by":"publisher","first-page":"7814","DOI":"10.1073\/pnas.79.24.7814","article-title":"Histone H2A subtypes associate interchangeably in vivo with histone H2B subtypes","volume":"79","author":"Kolodrubetz","year":"1982","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"9","key":"2025060406574381400_jkaf071-B21","doi-asserted-by":"publisher","first-page":"866","DOI":"10.1038\/s41592-019-0512-x","article-title":"An efficient auxin-inducible degron system with low basal degradation in human cells","volume":"16","author":"Li","year":"2019","journal-title":"Nat Methods"},{"key":"2025060406574381400_jkaf071-B22","doi-asserted-by":"publisher","first-page":"103364","DOI":"10.1016\/j.fgb.2020.103364","article-title":"Transformation of Cryptococcus neoformans by electroporation using a transient CRISPR-cas9 expression (TRACE) system","volume":"138","author":"Lin","year":"2020","journal-title":"Fungal Genet Biol"},{"issue":"5","key":"2025060406574381400_jkaf071-B23","doi-asserted-by":"publisher","first-page":"e0028323","DOI":"10.1128\/msphere.00283-23","article-title":"Rapid, efficient auxin-inducible protein degradation in Candida pathogens","volume":"8","author":"Milholland","year":"2023","journal-title":"mSphere"},{"issue":"2","key":"2025060406574381400_jkaf071-B24","doi-asserted-by":"publisher","first-page":"487","DOI":"10.1128\/EC.4.2.487-489.2005","article-title":"Thioredoxin reductase is essential for viability in the fungal pathogen Cryptococcus neoformans","volume":"4","author":"Missall","year":"2005","journal-title":"Eukaryot Cell"},{"issue":"2","key":"2025060406574381400_jkaf071-B25","doi-asserted-by":"publisher","first-page":"317","DOI":"10.1016\/j.femsle.2004.06.048","article-title":"Gene silencing with RNA interference in the human pathogenic fungus Aspergillus fumigatus","volume":"237","author":"Mouyna","year":"2004","journal-title":"FEMS Microbiol Lett"},{"issue":"10","key":"2025060406574381400_jkaf071-B26","doi-asserted-by":"publisher","first-page":"1299","DOI":"10.1017\/s1355838299990829","article-title":"Aberrant mRNAs with extended 3\u2032 UTRs are substrates for rapid degradation by mRNA surveillance","volume":"5","author":"Muhlrad","year":"1999","journal-title":"RNA"},{"issue":"5","key":"2025060406574381400_jkaf071-B27","doi-asserted-by":"publisher","first-page":"431","DOI":"10.1038\/s41589-018-0021-8","article-title":"The dTAG system for immediate and target-specific protein degradation","volume":"14","author":"Nabet","year":"2018","journal-title":"Nat Chem Biol"},{"key":"2025060406574381400_jkaf071-B28","doi-asserted-by":"publisher","author":"Nalley","year":"2025","DOI":"10.1101\/2025.02.14.638277"},{"issue":"12","key":"2025060406574381400_jkaf071-B29","doi-asserted-by":"publisher","first-page":"917","DOI":"10.1038\/nmeth.1401","article-title":"An auxin-based degron system for the rapid depletion of proteins in nonplant cells","volume":"6","author":"Nishimura","year":"2009","journal-title":"Nat Methods"},{"issue":"1","key":"2025060406574381400_jkaf071-B30","doi-asserted-by":"publisher","first-page":"6741","DOI":"10.1038\/ncomms7741","article-title":"Global analysis of fungal morphology exposes mechanisms of host cell escape","volume":"6","author":"O\u2019Meara","year":"2015","journal-title":"Nat Commun"},{"issue":"11","key":"2025060406574381400_jkaf071-B31","doi-asserted-by":"publisher","first-page":"919","DOI":"10.1002\/yea.1139","article-title":"An efficiently regulated promoter system for Cryptococcus neoformans utilizing the CTR4 promoter","volume":"21","author":"Ory","year":"2004","journal-title":"Yeast"},{"issue":"8","key":"2025060406574381400_jkaf071-B32","doi-asserted-by":"publisher","first-page":"1328","DOI":"10.1128\/EC.4.8.1328-1342.2005","article-title":"Tetracycline-inducible gene expression and gene deletion in Candida albicans","volume":"4","author":"Park","year":"2005","journal-title":"Eukaryot Cell"},{"issue":"11","key":"2025060406574381400_jkaf071-B33","doi-asserted-by":"publisher","first-page":"e1012735","DOI":"10.1371\/journal.ppat.1012735","article-title":"The Cryptococcus neoformans STRIPAK complex controls genome stability, sexual development, and virulence","volume":"20","author":"Peterson","year":"2024","journal-title":"PLoS Pathog"},{"issue":"12","key":"2025060406574381400_jkaf071-B34","doi-asserted-by":"publisher","first-page":"421","DOI":"10.3390\/biology9120421","article-title":"Targeted protein degradation tools: overview and future perspectives","volume":"9","author":"Prozzillo","year":"2020","journal-title":"Biology (Basel)"},{"issue":"12","key":"2025060406574381400_jkaf071-B35","doi-asserted-by":"publisher","first-page":"4153","DOI":"10.1073\/pnas.0400659101","article-title":"The plant hormone indoleacetic acid induces invasive growth in Saccharomyces cerevisiae","volume":"101","author":"Prusty","year":"2004","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"8","key":"2025060406574381400_jkaf071-B36","doi-asserted-by":"publisher","first-page":"873","DOI":"10.1016\/S1473-3099(17)30243-8","article-title":"Global burden of disease of HIV-associated cryptococcal meningitis: an updated analysis","volume":"17","author":"Rajasingham","year":"2017","journal-title":"Lancet Infect Dis"},{"issue":"29","key":"2025060406574381400_jkaf071-B37","doi-asserted-by":"publisher","first-page":"14698","DOI":"10.1073\/pnas.1902217116","article-title":"Predicting microbial growth in a mixed culture from growth curve data","volume":"116","author":"Ram","year":"2019","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"1","key":"2025060406574381400_jkaf071-B38","doi-asserted-by":"publisher","first-page":"153","DOI":"10.1111\/j.1365-2958.2004.04131.x","article-title":"RNA interference in Histoplasma capsulatum demonstrates a role for alpha-(1,3)-glucan in virulence","volume":"53","author":"Rappleye","year":"2004","journal-title":"Mol Microbiol"},{"issue":"1","key":"2025060406574381400_jkaf071-B39","doi-asserted-by":"publisher","first-page":"167","DOI":"10.1046\/j.1365-2958.2003.03697.x","article-title":"Large-scale essential gene identification in Candida albicans and applications to antifungal drug discovery","volume":"50","author":"Roemer","year":"2003","journal-title":"Mol Microbiol"},{"issue":"4","key":"2025060406574381400_jkaf071-B40","doi-asserted-by":"publisher","first-page":"e0064323","DOI":"10.1128\/msphere.00643-23","article-title":"Advanced genetic techniques in fungal pathogen research","volume":"9","author":"Ross","year":"2024","journal-title":"mSphere"},{"issue":"3","key":"2025060406574381400_jkaf071-B41","doi-asserted-by":"publisher","first-page":"507","DOI":"10.1016\/j.cell.2005.08.031","article-title":"Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile","volume":"123","author":"Schuldiner","year":"2005","journal-title":"Cell"},{"issue":"5","key":"2025060406574381400_jkaf071-B42","doi-asserted-by":"publisher","first-page":"e02048-18","DOI":"10.1128\/mBio.02048-18","article-title":"Gene essentiality analyzed by in vivo transposon mutagenesis and machine learning in a stable haploid isolate of Candida albicans","volume":"9","author":"Segal","year":"2018","journal-title":"mBio"},{"issue":"10","key":"2025060406574381400_jkaf071-B43","doi-asserted-by":"publisher","first-page":"jkac222","DOI":"10.1093\/g3journal\/jkac222","article-title":"The mIAA7 degron improves auxin-mediated degradation in Caenorhabditis elegans","volume":"12","author":"Sepers","year":"2022","journal-title":"G3 (Bethesda)"},{"issue":"1","key":"2025060406574381400_jkaf071-B44","doi-asserted-by":"publisher","first-page":"19","DOI":"10.1093\/genetics\/122.1.19","article-title":"A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae","volume":"122","author":"Sikorski","year":"1989","journal-title":"Genetics"},{"key":"2025060406574381400_jkaf071-B45","doi-asserted-by":"publisher","author":"Sladky","year":"2024","DOI":"10.1101\/2024.06.04.597287"},{"issue":"7909","key":"2025060406574381400_jkaf071-B46","doi-asserted-by":"publisher","first-page":"357","DOI":"10.1038\/s41586-022-04698-x","article-title":"CDC7-independent G1\/S transition revealed by targeted protein degradation","volume":"605","author":"Suski","year":"2022","journal-title":"Nature"},{"issue":"8","key":"2025060406574381400_jkaf071-B47","doi-asserted-by":"publisher","first-page":"567","DOI":"10.1002\/yea.3080","article-title":"iAID: an improved auxin-inducible degron system for the construction of a \u201ctight\u201d conditional mutant in the budding yeast Saccharomyces cerevisiae","volume":"32","author":"Tanaka","year":"2015","journal-title":"Yeast"},{"issue":"2","key":"2025060406574381400_jkaf071-B48","doi-asserted-by":"publisher","first-page":"444","DOI":"10.1128\/JB.181.2.444-453.1999","article-title":"A glucan synthase FKS1 homolog in Cryptococcus neoformans is single copy and encodes an essential function","volume":"181","author":"Thompson","year":"1999","journal-title":"J Bacteriol"},{"issue":"10","key":"2025060406574381400_jkaf071-B49","doi-asserted-by":"publisher","first-page":"767","DOI":"10.1038\/nmeth.1377","article-title":"Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms","volume":"6","author":"van Opijnen","year":"2009","journal-title":"Nat Methods"},{"key":"2025060406574381400_jkaf071-B50","doi-asserted-by":"publisher","first-page":"69","DOI":"10.1007\/978-1-0716-1720-5_4","article-title":"Design and generation of a CRISPR interference system for genetic repression and essential gene analysis in the fungal pathogen Candida albicans","volume":"2377","author":"Wensing","year":"2022","journal-title":"Methods Mol Biol"},{"issue":"8","key":"2025060406574381400_jkaf071-B51","doi-asserted-by":"publisher","first-page":"719","DOI":"10.1038\/nmeth.1231","article-title":"Yeast Barcoders: a chemogenomic application of a universal donor-strain collection carrying bar-code identifiers","volume":"5","author":"Yan","year":"2008","journal-title":"Nat Methods"},{"issue":"1","key":"2025060406574381400_jkaf071-B52","doi-asserted-by":"publisher","first-page":"5701","DOI":"10.1038\/s41467-020-19532-z","article-title":"The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice","volume":"11","author":"Yesbolatova","year":"2020","journal-title":"Nat Commun"}],"container-title":["G3: Genes, Genomes, Genetics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/g3journal\/advance-article-pdf\/doi\/10.1093\/g3journal\/jkaf071\/62885796\/jkaf071.pdf","content-type":"application\/pdf","content-version":"am","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/g3journal\/article-pdf\/15\/6\/jkaf071\/62885796\/jkaf071.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/g3journal\/article-pdf\/15\/6\/jkaf071\/62885796\/jkaf071.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,6,4]],"date-time":"2025-06-04T10:57:49Z","timestamp":1749034669000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/g3journal\/article\/doi\/10.1093\/g3journal\/jkaf071\/8107862"}},"subtitle":[],"editor":[{"given":"J","family":"Heitman","sequence":"additional","affiliation":[],"role":[{"role":"editor","vocabulary":"crossref"}]}],"short-title":[],"issued":{"date-parts":[[2025,4,7]]},"references-count":52,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2025,4,7]]},"published-print":{"date-parts":[[2025,6,4]]}},"URL":"https:\/\/doi.org\/10.1093\/g3journal\/jkaf071","relation":{},"ISSN":["2160-1836"],"issn-type":[{"value":"2160-1836","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2025,6]]},"published":{"date-parts":[[2025,4,7]]},"article-number":"jkaf071"}}