{"doi":"10.1101/530410","title":"Generation of conditional auxin-inducible degron (AID) cells and tight control of degron-fused proteins using the degradation inhibitor auxinole","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Controlling protein expression using a degron is drawing more attention because the protein of interest can be rapidly depleted in a reversible manner. We pioneered the development of the auxin-inducible degron (AID) technology by transplanting a plant-specific degradation pathway to non-plant cells. In human cells expressing an E3 ligase component, OsTIR1, it is possible to degrade a degron-fused protein with a half-life of 15–45 min in the presence of the phytohormone auxin. We reported previously the generation of human HCT116 mutants in which the C terminus of endogenous proteins was fused with the degron by CRISPR–Cas9-based knock-in. Here, we show new plasmids for N-terminal tagging and describe a detailed protocol for the generation of AID mutants of human HCT116 and DLD1 cells. Moreover, we report the use of an OsTIR1 inhibitor, auxinole, to suppress leaky degradation of degron-fused proteins. The addition of auxinole is also useful for rapid re-expression after depletion of degron-fused proteins. These improvements enhance the utility of AID technology for studying protein function in living human cells.</jats:p>","journal":null,"year":null,"id":592370,"datarank":0.6143941463201664,"base_score":2.8903717578961645,"endowment":2.8903717578961645,"self_citation_contribution":0.4335557636844247,"citation_network_contribution":0.18083838263574167,"self_endowment_contribution":0.4335557636844247,"citer_contribution":0.18083838263574167,"corpus_percentile":null,"corpus_rank":null,"citation_count":17,"citer_count":13,"citers_with_citation_signal":10,"citers_with_endowment":10,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":625850,"name":"Toyoaki Natsume","orcid":"0000-0002-3544-4491","position":1,"is_corresponding":false},{"id":1515767,"name":"Ken-ichiro Hayashi","orcid":null,"position":2,"is_corresponding":false},{"id":625852,"name":"Masato T. Kanemaki","orcid":"0000-0002-7657-1649","position":3,"is_corresponding":false},{"id":1515766,"name":"Aisha Yesbolatova","orcid":null,"position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"Generation of conditional auxin-inducible degron (AID) cells and tight control of degron-fused proteins using the degradation inhibitor auxinole","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>Controlling protein expression using a degron is drawing more attention because the protein of interest can be rapidly depleted in a reversible manner. We pioneered the development of the auxin-inducible degron (AID) technology by transplanting a plant-specific degradation pathway to non-plant cells. In human cells expressing an E3 ligase component, OsTIR1, it is possible to degrade a degron-fused protein with a half-life of 15–45 min in the presence of the phytohormone auxin. We reported previously the generation of human HCT116 mutants in which the C terminus of endogenous proteins was fused with the degron by CRISPR–Cas9-based knock-in. Here, we show new plasmids for N-terminal tagging and describe a detailed protocol for the generation of AID mutants of human HCT116 and DLD1 cells. Moreover, we report the use of an OsTIR1 inhibitor, auxinole, to suppress leaky degradation of degron-fused proteins. The addition of auxinole is also useful for rapid re-expression after depletion of degron-fused proteins. These improvements enhance the utility of AID technology for studying protein function in living human cells.</jats:p>","is_dataset_classified":null,"base_score":2.8903717578961645,"endowment":2.8903717578961645,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"26657633","pmcid":null,"openalex_id":"https://openalex.org/W2912616541","authors":[],"funders":[],"total_grants":0,"fwci":null,"citation_percentile":null,"influential_citations":0,"citation_trend":[{"year":2019,"count":4},{"year":2020,"count":1},{"year":2021,"count":2},{"year":2022,"count":5},{"year":2023,"count":2},{"year":2025,"count":3}],"oa_status":"green","license":null,"oa_locations":[{"url":"https://www.biorxiv.org/content/biorxiv/early/2019/04/08/530410.full.pdf","host_type":"repository"},{"url":"https://www.biorxiv.org/content/biorxiv/early/2019/04/08/530410.full.pdf","host_type":"repository"},{"url":"https://syndication.highwire.org/content/doi/10.1101/530410","host_type":"publisher"},{"url":"https://doi.org/10.1101/530410","host_type":"repository"}],"fields_of_study":["Ubiquitin and proteasome pathways","Protein Degradation and Inhibitors","CAR-T cell therapy research"],"mesh_terms":[],"keywords":["Degron","Mutant","Cell biology","Chemistry","Protein degradation","Bioproduction","Biochemistry","Biology","Ubiquitin ligase","Ubiquitin","Gene"],"sdg_mappings":[],"linked_datasets":[],"clinical_trials":[],"software_tools":[],"database_accessions":[],"source":"live","citation_network_status":"fetched"},"created_at":"2026-07-26T13:23:05.880866Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}