Generation of conditional auxin-inducible degron (AID) cells and tight control of degron-fused proteins using the degradation inhibitor auxinole
Aisha Yesbolatova, Toyoaki Natsume, Ken-ichiro Hayashi, Masato T. Kanemaki
Methods Available online 24 April 2019 DOI:10.1016/j.ymeth.2019.04.010
The auxin-inducible degron (AID) technology developed by the Kanemaki group is based on a plant-specific degradation pathway transplanted to non-plant cells. In human cells expressing a plant E3 ligase component, TIR1, a degron-fused endogenous protein is degraded within 15–45 min upon addition of the phytohormone auxin.
We demonstrated 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 (Natsume et al. Cell Reports, 2016). In this study, A SOKENDAI student, Aisha Yesbolatova, developed new plasmids for N-terminal tagging and described a detailed protocol for the generation of AID mutants of human HCT116 and DLD1 cells (Figure 1).
Figure1: (A) A new CRISPR/Cas9-based N-terminal tagging system. (B) C-terminal tagging.
Moreover, we report the use of a TIR1 inhibitor, auxinole, to suppress leaky degradation of degron-fused proteins (Figure 2). The addition of auxinole is also useful for rapid re-expression after depletion of degron-fused proteins.
Figure2: (A) The structure of auxin and auxinole, and illustrations showing how these reagents work in the degradation pathway. (B) Suppression of leaky degradation in DHC1-tagged Tet-TIR1 cells. The cells were treated with only doxycycline to induce TIR1 expression or together with auxinole 48 h before microscopy.
This study was carried out as a collaboration with Prof. Ken-ichiro Hayashi of Okayama University of Science and was supported by JSPS KAKENHI (17K15068, 18H02170 and 18H04719), JST A-STEP (grant number AS2915150U), the Canon Foundation, the Asahi Glass Foundation, and the Takeda Science Foundation.