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

Amit Rahi*, Deepika K. Sodhi, Christine B. Magdongon, Rajina Shakya, Dileep Varma*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Article numbere4923
JournalBio-protocol
Volume14
Issue number2
DOIs
StatePublished - Jan 20 2024

Keywords

  • Auxin-induced degradation
  • Auxin-inducible degron (AID)
  • CDT1
  • CRISPR/Cas9
  • Mitotic functions
  • Proteasomal degradation
  • Protein depletion
  • Protein regulation
  • Protein tagging
  • TIR1 receptor

ASJC Scopus subject areas

  • General Immunology and Microbiology
  • General Biochemistry, Genetics and Molecular Biology
  • General Neuroscience
  • Plant Science

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