{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T02:02:28Z","timestamp":1768528948550,"version":"3.49.0"},"posted":{"date-parts":[[2025,9,23]]},"group-title":"Cell Biology","reference-count":59,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,9,23]],"date-time":"2025-09-23T00:00:00Z","timestamp":1758585600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,9,23]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Targeted protein degradation is a favorable strategy for studying the immediate downstream effects of protein loss-of-function. An appealing platform among these technologies is the auxin-inducible degron (AID) system. Although this system has been applied extensively to cell and animal models, degradation resistance to long-term auxin treatment has not been studied. With the advent of the new AID2 system, cellular toxicity due to the high concentrations of auxin required in the original AID1 system is no longer a concern, making it possible to study protein degradation over extended periods. In this study, we derived multiple miniAID-tagged knock-in human cell lines and a Ctcf-miniAID knock-in mouse strain to investigate mechanisms of degradation resistance. We revealed four independent resistance mechanisms, including a nonsense mutation in the\n                  <jats:italic>CTCF<\/jats:italic>\n                  coding sequence that removed the miniAID peptide, a missense point mutation in the\n                  <jats:italic>miniAID<\/jats:italic>\n                  coding region that disrupted ubiquitin complex targeting, and silencing of the OsTIR1 adaptor protein. Resistance to auxin degradation was also acquired in mouse primary Ctcf\n                  <jats:sup>miniAID\/miniAID<\/jats:sup>\n                  knock-in B-ALL cells through missense mutations of the OsTIR1\n                  <jats:sup>(F74G)<\/jats:sup>\n                  protein\n                  <jats:italic>in vivo<\/jats:italic>\n                  and\n                  <jats:italic>ex vivo<\/jats:italic>\n                  . In summary, our innovative study expands our understanding of the AID system and cautions careful consideration of design for future applications in mammalian system.\n                <\/jats:p>","DOI":"10.1101\/2025.09.21.677607","type":"posted-content","created":{"date-parts":[[2025,9,23]],"date-time":"2025-09-23T13:05:15Z","timestamp":1758632715000},"source":"Crossref","is-referenced-by-count":0,"title":["Deciphering acquired resistance mechanisms to sustained auxin-inducible protein degradation in cells and 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