Methodology to Create Auxin-Inducible Degron Tagging System to Control Expression of a Target Protein in Mammalian Cell Lines

The auxin-inducible degron (AID) system is a versatile tool in cell biology and genetics, enabling conditional protein regulation through auxin-induced degradation. Integrating CRISPR/Cas9 with AID expedites tagging and depletion of a required protein in human and mouse cells. The mechanism of AID involves interactions between receptors like TIR1 and the AID tag fused to the target protein. The presence of auxin triggers protein ubiquitination, leading to proteasome-mediated degradation. We have used AID to explore the mitotic functions of the replication licensing protein CDT1. Swift CDT1 degradation via AID upon auxin addition achieves precise mitotic inhibition, revealing defects in mitotic spindle structure and chromosome misalignment. Using live imaging, we found that mitosis-specific degradation of CDT1 delayed progression and chromosome mis-segregation. AID-mediated CDT1 inhibition surpasses siRNA-based methods, offering a robust approach to probe CDT1’s mitotic roles. The advantages of AID include targeted degradation and temporal control, facilitating rapid induction and reversal of degradation—contrasting siRNA’s delayed RNA degradation and protein turnover. In summary, the AID technique enhances precision, control, and efficiency in studying protein function and regulation across diverse cellular contexts. In this article, we provide a step-by-step methodology for generating an efficient AID-tagging system, keeping in mind the important considerations that need to be adopted to use it for investigating or characterizing protein function in a temporally controlled manner.Key features• The auxin-inducible degron (AID) system serves as a versatile tool, enabling conditional protein regulation through auxin-induced degradation in cell biology and genetics.• Integration of CRISPR/Cas9 knock-in technology with AID expedites the tagging and depletion of essential proteins in mammalian cells.• AID’s application extends to exploring the mitotic functions of the replication licensing protein CDT1, achieving precise mitotic inhibition and revealing spindle defects and chromosome misalignment.• The AID system and its diverse applications advance the understanding of protein function and cellular processes, contributing to the study of protein regulation and function.Graphical overview Cdt1–auxin-inducible degron (AID) tagging workflow. (A) Schematic of the cloned Cdt1 gRNA vector and the repair template generated to endogenously tag the Cdt1 genomic locus with YFP and AID at the C-terminal using CRISPR/CAS9-based genome editing. The two plasmids are transfected into DLD1-TIR1 stable cells, followed by sorting and scaling up of YFP-positive single cells. (B) The molecular mechanism of auxin-induced proteasome-mediated degradation of the target protein (CDT1) shown at the bottom of the figure is well worked out.