  The ARF-AID (Auxin Response Factor-Auxin Inducible Degron) system is a
re-engineered auxin-inducible protein degradation system. Inducible degron
systems are widely used to specifically and rapidly deplete proteins of
interest in cell lines and organisms. An advantage of inducible degradation is
that the biological system under study remains intact and functional until
perturbation. This feature necessitates that the endogenous levels of the
protein are maintained. However, endogenous tagging of genes with AID can
result in chronic, auxin-independent proteasome-mediated degradation. The
additional expression of the ARF-PB1 domain in the re-engineered ARF-AID system
prevents chronic degradation of AID-tagged proteins while preserving rapid
degradation of tagged proteins. Here we describe the protocol for engineering
human cell lines to implement the ARF-AID system for specific and inducible
protein degradation. These methods are adaptable and can be extended from cell
lines to organisms.
