dTAG and aTAG Degraders
The dTAG and aTAG Degradation technologies offer a generalizable strategy to degrade, in principle, any intracellular protein of interest. The key benefit is that these techniques do not rely on the pre-existence of a ligand or PROTAC® for the protein of interest. The TAG Degradation Platform is a useful alternative to genetic knockdown/knockout methods for target validation and can be used in cell culture or in vivo.
Why Use dTAG and aTAG Degraders?
dTAG and aTAG Degraders can be used for target validation and exploration, offering an attractive alternative to genetic knockdown/knockout. The table below provides a comparison of TAG Degradation with commonly used genetic methods, including CRISPR/Cas9 and RNA interference. Key advantages of TAG Degraders include the ability to tune the extent of protein knockdown by varying the dose, and the more rapid onset of action (kinetics) for studying ‘fast biology’. In addition, dTAG and aTAG Degraders can be washed out of cell culture media, reversing their effect.
A Comparison of TAG Degradation with Genetic Methods of Protein Knockdown
|TAG Degradation Platforms (dTAG and aTAG)||***||****||****||***||****|
|Gene Knockout e.g. CRISPR/Cas||*||****||*||*||****|
|Gene Knockdown e.g. RNAi||*||***||*||*||****|
How do dTAG and aTAG Degraders Work?
The dTAG and aTAG platforms requires the protein of interest to be expressed as a fusion with a TAG protein via transgene expression or CRISPR-mediated locus-specific knock-in (see resources section below for custom TAG knock-in cell lines and protocols). Subsequent treatment with the relevant TAG Degrader targets the entire fusion protein for degradation. TAG Degraders mediate the formation of a ternary complex between an E3 ubiquitin ligase, such as cereblon (CRBN) or von Hippel Lindau (VHL) protein, and the fusion protein. This leads to polyubiquitination of the target protein and the subsequent degradation of the entire fusion protein by the proteasome. dTAG and aTAG Degraders act catalytically, repeatedly engaging and directing the ubiquitination of target molecules. They are cell-permeable and are suitable for in vitro and in vivo applications. The key difference between dTAG and aTAG technology is the identity of the TAG protein used.
The dTAG (or degradation TAG) system utilizes a single-point mutant FKBP12 (F36V) as the TAG domain with corresponding Degraders that selectively recruit FKBP12F36V over wild-type FKBP12. dTAG Degraders recruiting two different E3 ligases are available: dTAG-13 (Cat. No. 6605) and dTAG-7 (Cat. No. 6912) recruit CRBN while dTAGV-1 (Cat. No. 6914) recruits VHL. Negative controls are available.
The aTAG (Achilles TAG) system, uses the enzyme MTH1 (MutT homolog-1; NUDT1) as the TAG domain, which is expressed as a fusion with the protein of interest. MTH1 is used as the TAG in this system since the loss of this protein has no known phenotypic consequence resulting from the loss of this protein. aTAG Degraders, aTAG 2139 (Cat. No. 6970) and aTAG 4531 (Cat. No. 6971) comprise a ligand selective for MTH1 linked to a CRBN (E3 ligase) ligand and bring about potent and rapid degradation of the fusion protein by the proteasome.
PROTAC® is a registered trademark of Arvinas Operations, Inc., and is used under license.
Visit Bio-Techne brand Tocris to view the protocol for the CRISPR-mediated specific knock-in of MTH1-fusion proteins
Visit Addgene.org to find plasmid vectors for the lentiviral expression and CRISPR-mediated knock-in of FKBP12F36V
Bensimon, A et al. (2020) Targeted degradation of SLC transporters reveals amenability of multi-pass transmembrane proteins to ligand-induced proteolysis. Cell Chem.Biol. PMID: 32386596.
Nabet, B et al. (2018) The dTAG system for immediate and target-specific protein degradation. Nat. Chem. Biol. PMID: 29581585.
Nabet, B et al. (2020) Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules. Nat. Commun. PMID: 32948771.