Improved androgen specificity of AR-EcoScreen by CRISPR based glucocorticoid receptor knockout. (December 2017)

• Record Type: Journal Article
• Authors: Zwart, Nick; Andringa, Dave; de Leeuw, Willem-Jan; Kojima, Hiroyuki; Iida, Mitsuru; Houtman, Corine J.; de Boer, Jacob; Kool, Jeroen; Lamoree, Marja H.; Hamers, Timo
• Is Part Of: Toxicology in vitro. Volume 45:Part 1(2017)
• Page Start: 1
• Page End: 9
• Publication Date: 2017-12
• Subjects: AR androgen receptor -- ARE androgen responsive element -- CDTA 1, 2-cyclohexylenedinitrilotetraacetic acid -- CHO Chinese hamster ovary -- DBD DNA binding domain -- DSB double stranded breaks -- FACS fluorescent-activated cell sorting -- GC glucocorticoid -- GR glucocorticoid receptor -- GRE glucocorticoid responsive element -- HDR homology directed repair -- NHEJ non-homologous end-joining -- NR3C1 GR gene -- NTD N-terminal domain -- PAM Protospacer adjacent motif -- LBD ligand binding domain -- PS passive sampler -- SD speedisk -- sgRNA single guide RNA -- SNP single nucleotide polymorphism -- SR silicone rubber -- TU toxic units -- WWTP waste water treatment plant; CRISPR/Cas9 -- AR-EcoScreen -- Glucocorticoid receptor -- Knockout -- Bioassay; Toxicity testing -- In vitro -- Periodicals; Toxicology -- Periodicals; 615.9
• DOI: 10.1016/j.tiv.2017.08.004
• Languages: English
• ISSNs: 0887-2333

Abstract

Abstract: 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. Highlights: GR knockout renders the AR-EcoScreen assay insensitive to glucocorticoid activation. Sensitivity towards (anti-)androgens in mutants does not differ from AR-EcoScreen. AR-EcoScreen and mutants measure equal additivity in an androgenic mixture. Response to environmental mixtures differs between AR-EcoScreen and knockout mutants. GR knockout mutants detected specific androgen response in environmental mixtures.