https://www.nature.com/articles/s41589-018-0021-8

The dTAG system for immediate and target-specific protein degradation

Key figures

• [Fig. 1]: Establishes the core chemical-genetic mechanism—FKBP12F36V-selective engagement plus CRBN recruitment and ternary complex formation (including the “hook effect”).
• [Fig. 2]: Demonstrates rapid, selective, CRBN- and proteasome-dependent degradation in cells (dose + time-course + pathway dependency controls).
• [Fig. 3]: Shows endogenous locus control via FKBP12F36V knock-in (BRD4) enabling isoform-specific functional dissection versus pan-BET inhibition/degradation.
• [Fig. 6]: Validates rapid, reversible degradation in vivo using a luciferase-FKBP12F36V reporter and systemic dTAG-13 dosing.

1) Thesis (one sentence)

To address the gap of fast, target-specific, reversible control of single-protein abundance without requiring a bespoke target ligand, in mammalian cells and mice, FKBP12F36V tagging plus dTAG-13 treatment causes rapid allele-specific protein loss by CRBN-mediated ubiquitination and proteasomal degradation, supported by biochemical binding/dimerization assays and cellular/in vivo functional readouts.

2) Evidence card (three bullets only)

• Strongest result: (Fig. 2d) dTAG-13 drives rapid degradation of FKBP12F36V-tagged protein in cells within ~1 hour on a time course, consistent with immediate temporal control.
• Method enabler: (Fig. 1c–f; biochemical assay + tools) AlphaScreen ligand-displacement and CRBN-DDB1/FKBP12F36V proximity dimerization assays identify FKBP12F36V-selective, CRBN-recruiting degraders (dTAG-7/13) and quantify ternary behavior including a high-dose hook effect.
• Critical limitation: (Fig. 3b; Supplementary Fig. 4b–d) Homozygous C-terminal FKBP12F36V knock-in at BRD4 could not be recovered, implying tag placement can disrupt essential interaction domains (e.g., BRD4 C-terminal P-TEFb-binding region), constraining endogenous-tag design and interpretability.

Optional

Quote bank (2–4 short excerpts)

• Quote 1: “The dTAG system pairs a novel degrader of FKBP12F36V with expression of FKBP12F36V in-frame with a protein of interest.” (Abstract, p.1)
• Quote 2: “We show that ortho-substituted degraders dTAG-7 and dTAG-13 require CRBN to induce degradation, and are highly selective for FKBP12F36V.” (Discussion, p.9)
• Quote 3: “No significantly differentially expressed genes were identified upon dTAG-13 treatment, further validating the high degree of selectivity of dTAG-13.” (KRASG12V transcriptional signaling section, p.6)

Key comparisons (1–3 lines)

• Compared to: AID / HaloTag(-PROTAC) / SMASh systems that require exogenous machinery, alternative tags, or different degron logic.
• Win: Uses endogenous CRBN machinery (no added E3 component) and achieves rapid, allele-selective loss with a broadly reusable FKBP12F36V handle.
• Tradeoff: Requires engineering (fusion/knock-in) and exhibits ternary “hook effect” dose behavior plus tag-position sensitivity (e.g., BRD4 C-terminal constraints).

Methods I might copy (protocol hooks)

• Construct design / Models: FKBP12F36V N- or C-terminal fusion via Gateway-compatible lentiviral pLEX_305 N-dTAG / C-dTAG; PITCh (MMEJ) CRISPR strategy for locus knock-in (BRD4); luciferase-FKBP12F36V reporter for in vivo readout.
• Conditions / Instruments: Degradation observed at 50–100 nM dTAG-7/13 in some cellular contexts and on ~1 h timescales for several fusions; cultured lines include MV4;11, NIH/3T3, 293T, 293FT at 37 °C and 5% CO2; Envision 2104 plate reader for luminescence AlphaScreen/dual-luciferase; Odyssey CLx for IR immunoblot imaging.
• Readout / Analysis: Dual-luciferase Nluc/Fluc degradation quantification; immunoblot pathway readouts (e.g., pMEK S221, pAKT S473); multiplexed TMT LC-MS3 proteomics/phosphoproteomics (Orbitrap Fusion/Lumos); RNA-seq with ERCC spike-in normalization (NextSeq 500) plus GSEA/IPA.

Open questions / Theoretical implications (2–5 bullets)

• How predictable is degradation kinetics from target subcellular localization, complex membership, or basal turnover (e.g., KRASG12V fusion slower than several nuclear targets in the same framework)?
• What design rules best minimize the hook effect window while preserving cell permeability and CRBN engagement across targets?
• Can alternative E3 ligase recruiters (beyond CRBN) reduce neosubstrate liabilities while preserving “universal tag” generalizability?
• How often does endogenous knock-in fail because of functional disruption at the tag junction, and can linker/placement heuristics be standardized?