{"doi":"10.1101/2025.01.21.633519","title":"A single-chain antibody-based AID2 system for conditional degradation of GFP-tagged and untagged proteins","abstract":"Abstract Protein knockdown using an improved auxin-inducible degron (AID2) technology has proven to be a powerful tool for studying protein function. The current approach requires the fusion of target proteins with a degron tag, a process typically achieved through CRISPR knock-in. However, knock-in remains challenging in non-model organisms and humans, limiting the broader applicability of AID2. To overcome this limitation, we developed a single-chain antibody AID2 (scAb-AID2) system. This approach employs an adaptor composed of a single-chain antibody fused with a degron, which recognises a target protein and induces rapid degradation in the presence of the inducer 5-Ph-IAA. We demonstrated that scAb-AID2, in combination with an anti-GFP nanobody, degraded GFP-fused proteins in human cells and C. elegans . Furthermore, we showed that endogenous p53 and H/K-RAS were conditionally degraded in cells expressing an adaptor encoding an anti-p53 nanobody and -RAS monobody, respectively, and led to aphidicolin sensitivity in cell culture and growth inhibition in mouse xenografts. This study paves the way for broader application of AID2-based target depletion in model and non-model organisms and for advancing therapeutic strategies.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":560818,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9535,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":588354,"name":"Takefumi Negishi","orcid":"0000-0002-0306-9578","position":1,"is_corresponding":false},{"id":1463187,"name":"Naomi Kitamoto","orcid":null,"position":2,"is_corresponding":false},{"id":1425647,"name":"Yuki Hatoyama","orcid":"0000-0001-5554-8311","position":3,"is_corresponding":false},{"id":1463188,"name":"Kanae Gamo","orcid":null,"position":4,"is_corresponding":false},{"id":458492,"name":"Ken‐ichiro Hayashi","orcid":"0000-0002-9812-2801","position":5,"is_corresponding":false},{"id":625852,"name":"Masato T. Kanemaki","orcid":"0000-0002-7657-1649","position":6,"is_corresponding":false},{"id":1462869,"name":"Moutushi Islam","orcid":"0000-0002-0934-9247","position":0,"is_corresponding":true}],"reference_count":66,"raw_metadata":null,"created_at":"2026-07-19T02:55:46.921885Z","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":[]}