Generation of conditional auxin-inducible degron (AID) cells and tight control of degron-fused proteins using the degradation inhibitor auxinole

•The manuscript shows our protocol to generate conditional human cell lines using the auxin-inducible degron technology.•CRISPR–Cas9-based gene tagging is used.•New donor plasmids offer to fuse the degron or other tags at the N-terminus of a protein of interest.•New degradation inhibitor auxinole can suppress leaky degradation of degron-fused proteins in the absence of auxin.•Auxinole can be used to achieve rapid recovery of degron-fused proteins after auxin removal. Controlling protein expression using a degron is advantageous 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.