An efficient auxin-inducible degron system with low basal degradation in human cells

Abstract

Auxin-inducible degron technology allows rapid and controlled protein depletion. However, basal degradation without auxin and inefficient auxin-inducible depletion have limited its utility. We have identified a potent auxin-inducible degron system composed of auxin receptor F-box protein AtAFB2 and short degron miniIAA7. The system showed minimal basal degradation and enabled rapid auxin-inducible depletion of endogenous human transmembrane, cytoplasmic and nuclear proteins in 1 h with robust functional phenotypes. Loss-of-function methods for reducing target protein levels target DNA, RNA or protein 1. Auxin-inducible degron (AID) technology allows rapid targeted protein depletion with the smallmolecule auxin 2. To apply AID, an auxin-inducible destabilizing domain, or 'degron' , is fused to the target protein. In addition, an auxin receptor F-box protein TIR1/AFB is exogenously expressed, forming a chimeric SKP1-CUL1-F-Box (SCF) ubiquitin E3 ligase with endogenous components in eukaryotic cells. The chimeric E3 ligase recruits the degron in an auxin-dependent manner 3. Addition of indole-3-acetic acid (IAA) or other small molecules of the auxin class to the culture medium thus induces the polyubiquitination and rapid proteasomal degradation of the degron-fused protein (Fig. 1a). This approach has been used as a powerful tool to acutely deplete target proteins in studying their functions 4-7. However, current AID systems can severely degrade target proteins before IAA addition (known as 'basal degradation') and suffer from inefficient auxin-inducible depletion in a context-and target-specific manner 2,8-10. These pitfalls have substantially limited the applications of AID technology. An AID system with both minimal basal degradation and rapid auxin-inducible depletion would be desirable. In current AID systems, Oryza sativa TIR1 (OsTIR1) is the most commonly used auxin receptor F-box protein in combination with different degrons deriving from Arabidopsis thaliana IAA17 (AtIAA17) 2,4,5,7-14. We initially aimed to acutely deplete endogenous human seipin, a transmembrane protein involved in lipid droplet (LD) biogenesis. However, seipin tagged with a degron termed miniAID (composed of AtIAA17 amino acid (aa) 68-132) was severely degraded in cells expressing OsTIR1 without IAA addition. Consequently, cells exhibited defective LD biogenesis already before IAA addition, resembling a seipin knockout phenotype 15 (Supplementary Fig. 1a-c). To search for an improved AID system, we established a pipeline in human A431 cells for screening various auxin receptor F-box proteins and degrons, through co-integration into the adenoassociated virus integration site 1 (AAVS1) safe harbor locus 16 (Fig. 1b and Supplementary Note 1). We first compared OsTIR1 to five other auxin receptor F-box proteins, using miniAID as the degron. A. thaliana AFB2 (AtAFB2) was identified as the best hit: compared to OsTIR1, it displayed minimal basal depletion

Rapid Degradation ofCaenorhabditis elegansProteins at Single-Cell Resolution with a Synthetic Auxin

“…While we are optimistic that the use of the synthetic analog of auxin presented here will allow even more widespread utility of the AID system in the C. elegans research community, there are still some areas open to improvement for the technology. Recent reports in mammalian cell culture identified that AID-tagged proteins are depleted in an auxin-independent fashion in the presence of TIR1, relative to wild-type levels (Natsume et al 2016;Li et al 2019a;Sathyan et al 2019). We examined if this was also occurring in C. elegans strains in our laboratory expressing AID-tagged proteins and TIR1.…”

Ancient MAPK ERK7 is regulated by an unusual inhibitory scaffold required for Toxoplasma apical complex biogenesis

“…We assessed the basal (-IAA) level of AC9 AID protein, and found that AC9 AID levels are ~40% of the levels of AC9 tagged with only a 3xHA in the same OsTIR1-expressing parental line ( Figure S1). Such basal degradation has been uncovered as a common artifact of the AID system (30,31). Notably, AC9 AID (wtcomp) conoids were indistinguishable from the parental strain, suggesting that reduced AC9 protein levels may be driving the apparent defect in conoid ultrastructure (Figures 1, S1).…”

An endoribonuclease-based feedforward controller for decoupling resource-limited genetic modules in mammalian cells

“…In systems were relative DNA dosages cannot be easily tuned, RNA aptazymes 41 and/or inducible protein degradation domains [42][43][44] could be used to independently tune output levels. To control multiple output genes, it will be possible to construct additional orthogonal iFFLs using our recently-described panel of endoRNases 32 .…”