Universal donor plasmids for biallelic degron-tagging in mammalian cells and organoids
Pohl, S. O., Hernandez-Moran, B. A., Guzman Cardoso, C., Giller, A., Tayor, G. C., Myant, K., Macias, S., Wood, A. J.
synthetic biology
Quick verdict
Universal CRISPaint donors enable biallelic degron tagging in cells and organoids; useful method advance, though only few loci validated and stats are thin.
Summary
This method paper from the University of Edinburgh adapts the CRISPaint homology-independent knock-in system into a panel of universal donor plasmids for biallelic C-terminal degron tagging of endogenous genes in mammalian cells and organoids. Donors carry two frame-selector spacer sites so a single plasmid series — dTAG with mClover3/mScarlet-i3 fluorescent markers (direct or P2A-liberated, leaving a ~15 kDa tag), dTAG with hygromycin/puromycin resistance, AID2 with eGFP plus puromycin/blasticidin, and schematic BromoTag/SMASh versions — can be applied to any C-terminal locus chosen from a published target-selector sgRNA table. Proof-of-principle spans human Jurkat T cells (RASA2, SNX9, EZH2, TCF7), mouse embryonic stem cells (Dcp2; 12/37 single-cell clones biallelic, ~85% dTAG-13 depletion maintained over 72 h), and AKPS colorectal tumour organoids (Srsf1; rapid 5-Ph-IAA-dependent loss within 4 h, p<0.0001). A functional RASA2 assay confirms the expected PD-1 increase upon degradation but also shows large C-terminal fusions perturb function even uninduced, motivating the P2A format. Main limitations: validation is narrow (essentially one locus per selection modality; BromoTag/SMASh unvalidated in the presented data), genomic QC relies on junction PCR/Sanger without copy-number or off-target checks, and statistical reporting (n, pseudoreplication in Fig 3H) is thin.
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Assessment at a glance
4
risk flags identified
● high 0
● medium 3
● low 1
5 strengths · 5 weaknesses · 5 author questions
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Key claims under review
- A universal CRISPaint donor series enables biallelic, in-frame C-terminal integration of inducible degron tags at any chosen endogenous locus without constructing new donors per gene.
- Pairing two orthogonal selection markers (fluorescent FACS gates or dual antibiotic resistances) efficiently recovers biallelic knock-in cells (32% of mESC single-cell clones at Dcp2).
- Tagged proteins degrade rapidly and deeply upon ligand addition (DCP2 ~70% loss at 2 h with 100 nM dTAG-13 and >90% by 8 h; SRSF1 loss within 4 h of 200 nM 5-Ph-IAA in organoids).
- The workflow succeeds in contexts where homology-directed repair is challenging, including mouse colonic tumour organoids.
- Large C-terminal fusions can perturb endogenous protein function even without degrader induction (RASA2/PD-1), motivating the P2A small-tag donor format.
Strengths
True universality: no per-locus donor cloning
Frame-selector spacer sites are built into the donors, so any C-terminal locus from the published Schmid-Burgk target-selector sgRNA table (ref. 19) can be tagged with the same plasmid series, including a generic 3'UTR.
Orthogonal selection directly enriches biallelic events
Pairs of markers (mClover3/mScarlet-i3 for FACS; HygroR/PuroR in mESCs; PuroR/BlastR sequential rounds in organoids) yield 32% biallelic on-target clones among 37 single-cell mESC clones at Dcp2 (Fig. 2D), a concrete efficiency benchmark.
P2A design addresses tag-size perturbation, backed by their own data
Validation in three biologically distinct, increasingly demanding models
Strong wet-lab reproducibility package
Weaknesses
Validation breadth lags the abstract's four-tag claim
Genomic quality control is minimal
Thin statistical reporting throughout
One-locus-per-modality evidence for the antibiotic route
Citation integrity slip
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Scope & Claims
This is a methods/resource preprint (bioRxiv, posted 17 September 2026) from the Wood, Macias and Myant laboratories at the University of Edinburgh. It presents a panel of universal CRISPaint donor plasmids for biallelic, in-frame C-terminal fusion of inducible degron tags — dTAG (FKBP12F36V), AID2, plus constructed but largely unvalidated BromoTag and SMASh donors — to endogenous genes without locus-specific donor cloning, using dual fluorescent selection (mClover3/mScarlet-i3 FACS) or dual antibiotic selection (HygroR/PuroR; PuroR/BlastR). Three claims structure the paper: (1) the donor series is universal — any C-terminal locus can be targeted using the published Schmid-Burgk target-selector sgRNA table (ref. 19); (2) orthogonal markers enrich biallelic knock-ins; (3) the workflow works in human T cells, mouse ESCs and colorectal tumour organoids, including HDR-challenging contexts. The lineage is credited honestly: CRISPaint (ref. 19), universal fluorescent donors in mESCs (ref. 31), organoid NHEJ tagging (ref. 20), HiHo-AID2 (ref. 28). The contribution is packaging these into a biallelic, multi-degron resource, not inventing universal donors.
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Frequently asked questions
What does this paper introduce?
A set of universal donor plasmids that let researchers fuse inducible degron tags (dTAG, AID2, plus constructed BromoTag and SMASh donors) onto the C-terminus of a chosen gene in mammalian cells and organoids, and isolate cells in which both gene copies are tagged — without building a new donor vector for each gene.
How can both alleles be tagged without homology-directed repair?
The method adapts CRISPaint: a target-selector guide RNA cuts just upstream of the gene's stop codon, and a frame-selector guide linearizes the donor plasmid in the matching reading frame; the cell's NHEJ repair pathway stitches the donor into the cut. Two different selection markers (fluorescent proteins for FACS, or two antibiotic-resistance genes) let researchers enrich cells where both alleles carry a tag.
How efficient is the system and how fast is degradation?
In mouse embryonic stem cells at the Dcp2 locus, 32% of 37 single-cell clones were biallelic on-target knock-ins (45% monoallelic). Degradation is rapid: DCP2 lost about 70% within 2 hours of 100 nM dTAG-13 (over 90% by 8 hours), and SRSF1 in colorectal tumour organoids degraded within 4 hours of 200 nM 5-Ph-IAA.
Where can the plasmids and guide sequences be obtained?
The donor plasmids have been submitted to Addgene (submission ID 87793). Target-selector sgRNA sequences for human and mouse C-terminal tagging come from the previously published CRISPaint sgRNA table (Schmid-Burgk et al. 2016); the paper also lists screening primers and culture/editing conditions.
About this review
This is an AI-generated preliminary review by Evolve.Pub, produced from the openly available text of the paper. It is not peer review and does not represent the authors' views. The canonical record of the paper is at the publisher link above.
- Extracted text is heavily truncated: part of the Jurkat Results (Fig. 1D efficiency values), organoid kinetics details and the entire Discussion are missing.
- Figures are available only as embedded text legends; blots, flow plots and microscopy could not be inspected.
- BromoTag/SMASh validation data may exist in omitted sections; the unvalidated judgment is based on the visible text only.
- Data availability statements are not visible in the extraction; only the Addgene plasmid submission (ID 87793) is stated.
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