Universal donor plasmids for biallelic degron-tagging in mammalian cells and organoids

Abstract

Degron tags enable rapid, tuneable and reversible control of protein expression, and have become a critical tool for target validation and elucidating biological mechanisms. Tag engineering uses genome editing to generate biallelic gene fusions, though this process remains laborious and requires new donor vectors to be generated for each locus. Here, we have generated a series of universal donor vectors to enable biallelic integration of different degron tags (AID, dTAG, BromoTag, SMASh) using different selection strategies (FACS, drug selection). Using this system, tags can be fused in-frame to endogenous protein C-termini via homology-independent repair following Cas9-induced double strand breaks, as shown here using human lymphoma cells, mouse embryonic stem cells and colonic tumour organoids. This workflow simplifies the genetic engineering of degron tags and is applicable in contexts where homology-dependent repair is challenging.

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BibTeX RIS

BibTeX

@misc{pohl2026universal,
  title = {{Universal donor plasmids for biallelic degron-tagging in mammalian cells and organoids}},
  author = {S. O. Pohl and B. A. Hernandez-Moran and C. Guzman Cardoso and A. Giller and G. C. Tayor and K. Myant and S. Macias and A. J. Wood},
  howpublished = {bioRxiv},
  year = {2026},
  month = sep,
  doi = {10.64898/2026.09.14.751494},
  url = {https://www.biorxiv.org/content/10.64898/2026.09.14.751494v1},
  note = {CC BY-NC},
}

RIS

TY  - UNPB
TI  - Universal donor plasmids for biallelic degron-tagging in mammalian cells and organoids
AU  - S. O. Pohl
AU  - B. A. Hernandez-Moran
AU  - C. Guzman Cardoso
AU  - A. Giller
AU  - G. C. Tayor
AU  - K. Myant
AU  - S. Macias
AU  - A. J. Wood
PY  - 2026
DA  - 2026/09/14
PB  - bioRxiv
DO  - 10.64898/2026.09.14.751494
UR  - https://www.biorxiv.org/content/10.64898/2026.09.14.751494v1
AB  - Degron tags enable rapid, tuneable and reversible control of protein expression, and have become a critical tool for target validation and elucidating biological mechanisms. Tag engineering uses genome editing to generate biallelic gene fusions, though this process remains laborious and requires new donor vectors to be generated for each locus. Here, we have generated a series of universal donor vectors to enable biallelic integration of different degron tags (AID, dTAG, BromoTag, SMASh) using different selection strategies (FACS, drug selection). Using this system, tags can be fused in-frame to endogenous protein C-termini via homology-independent repair following Cas9-induced double strand breaks, as shown here using human lymphoma cells, mouse embryonic stem cells and colonic tumour organoids. This workflow simplifies the genetic engineering of degron tags and is applicable in contexts where homology-dependent repair is challenging.
ER  -

Abstract

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