ARF‐AID: A Rapidly Inducible Protein Degradation System That Preserves Basal Endogenous Protein Levels

Abstract

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, a feature that necessitates that the endogenous levels of the protein are maintained. However, endogenous tagging of genes with auxin-inducible degrons (AID) can result in chronic, auxin-independent proteasome-mediated degradation. The ARF-AID (auxin-response factorauxin-inducible degron) system is a re-engineered auxin-inducible protein degradation system. The additional expression of the ARF-PB1 domain prevents chronic, auxin-independent degradation of AID-tagged proteins while preserving rapid auxin-induced 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.

Evaluation of the OsTIR1 and AtAFB2 AID systems for chromatin protein degradation in mammalian cells

“…It is well known, that random integration of transgene leads to variable expression due to position effects and silencing. Integration of the OsTIR1 and AtAFB2 transgenes in AAVS1 safe-harbor locus in human cells is considered a sufficient measure to provide stable transgene expression [3,10,12]. We inserted AtAFB2 transgene into AAVS1 locus in one of the HAP1 line with Rad21-miniIAA7-eGFP modification.…”

“…4D). We feel that this fact is either omitted or not stressed enough in most published protocols [3,9,11,12]. Our observations also warn against removing selection cassette after AFB integration in safe-harbor loci [7].…”

“…Recent study reports that basal degradation in OsTIR1 AID could be countered by overexpressing ARF protein (Sathyan et al, 2020). Unfortunately, this approach is not applicable for the popular miniAID (mAID) degron system, because mAID lacks ARF-interacting domains [12].…”

“…During last years, researchers have come up with several strategies to generate stable transgenic cell lines expressing AID system. But even though many great protocols exist [9,11,12], the method still contains pitfalls that could obstruct new users. For instance, selection of the proper transgenic clone with high POI degradation efficiency could be unmanageable due to AFB silencing.…”

Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells

“…During last years, researchers have come up with several strategies to generate stable transgenic cell lines expressing the AID system. But even though many great protocols exist ( Lambrus et al, 2018 ; Yesbolatova et al, 2019 ; Sathyan et al, 2020 ), the method still contains pitfalls that could obstruct new users. For instance, selection of the proper transgenic clone with high POI degradation efficiency could be unmanageable due to AFB silencing.…”