The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice

By Aisha Yesbolatova, Yuichiro Saito, N. Kitamoto, Hatsune Makino-Itou, R. Ajima, Risako Nakano, H. Nakaoka, Kosuke Fukui, K. Gamo, Yusuke Tominari and 4 more. 2020

398 citations recorded by the source corpus.

Protein knockdown using the auxin-inducible degron (AID) technology is useful to study protein function in living cells because it induces rapid depletion, which makes it possible to observe an immediate phenotype. However, the current AID system has two major drawbacks: leaky degradation and the requirement for a high dose of auxin. These negative features make it difficult to control precisely the expression level of a protein of interest in living cells and to apply this method to mice. Here, we overcome these problems by taking advantage of a bump-and-hole approach to establish the AID version 2 (AID2) system. AID2, which employs an OsTIR1(F74G) mutant and a ligand, 5-Ph-IAA, shows no detectable leaky degradation, requires a 670-times lower ligand concentration, and achieves even quicker degradation than the conventional AID. We demonstrate successful generation of human cell mutants for genes that were previously difficult to deal with, and show that AID2 achieves rapid target depletion not only in yeast and mammalian cells, but also in mice. Auxin-inducible degron systems can be leaky and require high doses of auxin. Here the authors establish AID2 which uses an OsTIR1 mutant and the ligand 5-Ph-IAA to overcome these problems and establish AID-mediated target depletion in mice.

Subfields: Molecular Biology, Oncology

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In Molecular Biology

1. Yuichiro Saito
2. Masato T. Kanemaki
3. Johannes C. Walter
4. Sachiko Muramatsu

In Oncology

1. Yuichiro Saito
2. Masato T. Kanemaki

Citations in the corpus

• In silico protein interaction screening uncovers DONSON’s role in replication initiation (2023, methodology): “…was transfected with two CRISPR plasmids targeting the C-terminal coding region of the DONSON gene (targets: 5 ′ -TTAGGCTTACTT- TGGTGTTC-3 ′ , 5 ′ -TTAGGCTTACTTTGATGTTC- 3 ′ ) and a donor plasmid encoding mAID-mClover and a hygromycin resistant marker following a published protocol ( 38 , 68 ).”
• MCMBP promotes the assembly of the MCM2-7 hetero-hexamer to ensure robust DNA replication in human cells (2022, methodology): “MCMBP depletion causes loss of MCM3 and MCM5 from the MCM2-7 hexamer To investigate the effect of MCMBP depletion on the MCM2-7 hexamer, we generated a conditional mutant using the AID2 system (Yesbolatova et al., 2020).”