Systematic profiling of conditional degron tag technologies for target validation studies

Abstract

Conditional degron tags (CDTs) are a powerful tool for target validation that combines the kinetics and reversible action of pharmacological agents with the generalizability of genetic manipulation. However, successful design of a CDT fusion protein often requires a prolonged, ad hoc cycle of construct design, failure, and re-design. To address this limitation, we report here a system to rapidly compare the activity of five unique CDTs: AID/AID2, IKZF3d, dTAG, HaloTag, and SMASh. We demonstrate the utility of this system against 16 unique protein targets. We find that expression and degradation are highly dependent on the specific CDT, the construct design, and the target. None of the CDTs leads to efficient expression and/or degradation across all targets; however, our systematic approach enables the identification of at least one optimal CDT fusion for each target. To enable the adoption of CDT strategies more broadly, we have made these reagents, and a detailed protocol, available as a community resource.

Combination of AID2 and BromoTag expands the utility of degron-based protein knockdowns

“…S1A ) (Bond et al ., 2021; Nabet et al ., 2020; Nabet et al ., 2018; Nowak et al ., 2021). Even though AID2, dTAG and BromoTag have been used in cell biology, there are only a few studies comparing these systems (Bondeson et al ., 2022; Noviello et al ., 2023). To understand the similarities and differences among AID2, dTAG and BromoTag systems, we constructed a GFP reporter containing the three degrons in tandem ( Fig.…”

“…Both dTAG and BromoTag have been used in many studies, indicating that they are also promising conditional degrons (Evrin et al , 2023; Mylonas et al , 2021; Olsen et al , 2022; Weintraub et al , 2017). However, few studies have compared conditional degrons to discern their similarities and differences, and BromoTag has not yet been included for comparison (Bondeson et al , 2022; Noviello et al , 2023). Furthermore, the target proteins fused with each degron in these studies were overexpressed to varying levels, impacting degradation kinetics and complicating the comparison.…”

Continuous evolution of compact protein degradation tags regulated by selective molecular glues

“…Technologies for small molecule-induced degradation of target proteins include the degradation tag (dTAG) system (17,18), small moleculeassisted shutoff (SMASh) (19), ligand-induced degradation (20), HaloProtacs (21,22), and the auxin-inducible degron (23)(24)(25). These tags, however, are primarily limited to exogenous expression of fusion proteins owing to their large size (108 to 304 amino acids) or require coexpression of non-native effector domains (23,26). This requirement precludes the study of proteins expressed from their endogenous genomic loci at native expression levels under endogenous regulatory control.…”

Orthogonal IMiD-Degron Pairs Induce Selective Protein Degradation in Cells

“…This definition encompasses inducible degrons, which require the presence of a small molecule to promote protein degradation. Genetic knock-in methods enable a degron to be used in a complementary manner to PROTACs, allowing induced degradation of POIs that lack a small molecule ligand (25,28). A number of these technologies are well established, including the auxin inducible degron system (AID), small molecule assisted shutoff (SMASh-tag), a destabilizing domain (DD) stabilized by small molecule Shld1, a degron based on methyl guanine methyltransferase (MGMT), and systems that utilize bifunctional small molecules, including dTAG (degron = 11.9 kDa), HaloPROTAC (degron = 33.6 kDa), Bromotag (degron = 14.9 kDa), and an approach based on NanoLuciferase (28)(29)(30)(31)(32)(33)(34)(35)(36)(37).…”

“…A number of these technologies are well established, including the auxin inducible degron system (AID), small molecule assisted shutoff (SMASh-tag), a destabilizing domain (DD) stabilized by small molecule Shld1, a degron based on methyl guanine methyltransferase (MGMT), and systems that utilize bifunctional small molecules, including dTAG (degron = 11.9 kDa), HaloPROTAC (degron = 33.6 kDa), Bromotag (degron = 14.9 kDa), and an approach based on NanoLuciferase (28)(29)(30)(31)(32)(33)(34)(35)(36)(37). While these techniques have been elegantly applied in a numerous studies, a potential drawback is the use of large degron motifs, which can negatively affect feasibility of CRISPR knock-in and function of the POI (25,34). Recent work by Tsang et al offers a potential solution to this using HiBiT-SpyTag and SpyCatcher to add a degron motif to a POI (38).…”