Improved androgen specificity of AR-EcoScreen by CRISPR based glucocorticoid receptor knockout

Nick Zwart*, Dave Andringa, Willem Jan de Leeuw, Hiroyuki Kojima, Mitsuru Iida, Corine J. Houtman, Jacob de Boer, Jeroen Kool, Marja H. Lamoree, Timo Hamers

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review


The AR-EcoScreen is a widely used reporter assay for the detection of androgens and anti-androgens. Endogenous expression of glucocorticoid receptors and their affinity for the androgen responsive element that drives reporter expression, however, makes the reporter cells sensitive to interference by glucocorticoids and less specific for (anti-)androgens. To create a glucocorticoid insensitive derivative of the AR-EcoScreen, CRISPR/Cas9 genome editing was used to develop glucocorticoid receptor knockout mutants by targeting various sites in the glucocorticoid gene. Two mutant cell lines were further characterized and validated against the unmodified AR-EcoScreen with a set of 19 environmentally relevant chemicals and a series of environmental passive sampler extracts with (anti-)androgenic activity. Sequencing of the targeted sites revealed premature stop codons following frame-shift mutations, leading to an absence of functional glucocorticoid receptor expression. The introduced mutations rendered cell lines insensitive to glucocorticoid activation and caused no significant difference in the responsiveness towards (anti-)androgens, compared to the unmodified AR-EcoScreen cells, allowing the selective, GR-independent, determination of (anti-)androgenicity in environmental passive sampler extracts. The increase in selectivity for (anti-)androgens improves reliability of the AR-EcoScreen and will provide higher accuracy in determining (anti-)androgenic potential when applied in toxicity screening and environmental monitoring of both single compounds and mixtures.

Original languageEnglish
Pages (from-to)1-9
Number of pages9
JournalToxicology in Vitro
Publication statusPublished - 1 Dec 2017


The developed mutant 1 cell line will be deposited for distribution at the Japanese Collection of Research Bioresources (JCRB) Cell Bank ( ) under the name “AR-EcoScreen GR KO M1”. This research was funded by the Dutch Technology Foundation (STW), project number 12396 . The authors acknowledge Dr. Feng Zhang (Massachusetts Institute of Technology, Cambridge, MA, USA) for providing the pSpCas9(BB)-2A-GFP plasmid, Marjo den Broeder (Brunel University, London, United Kingdom) for assistance with experimental design and Tom O'Toole (Vrije Universiteit Medical Center, Amsterdam, The Netherlands) for assistance with FACS. Passive samplers were obtained within the CEFIC-LRI ECO-23 funded TIPTOP project.

FundersFunder number
Massachusetts Institute of Technology
London Deanery
Japan Society for the Promotion of Science15H04780, 15K08791
Stichting voor de Technische Wetenschappen
Vrije Universiteit Brussel
Arab Science and Technology Foundation12396


    • AR-EcoScreen
    • Bioassay
    • CRISPR/Cas9
    • Glucocorticoid receptor
    • Knockout


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