Science Inventory

Functional Genomic-Based Characterization of the Retinoic Acid Pathway in U-2 OS Cells Using CRISPRa-dCas9

Citation:

O'Neill, J., J. Harrill, AND B. Chorley. Functional Genomic-Based Characterization of the Retinoic Acid Pathway in U-2 OS Cells Using CRISPRa-dCas9. 2024 SOT Annual Meeting, Salt Lake City, UT, March 10 - 14, 2024. https://doi.org/10.23645/epacomptox.25533442

Impact/Purpose:

Abstract submitted for SOT 2024 to support ORISE Jennifer O'Neill. Abstract describe efforts to utilize CRISPR-based induction of retinoic acid pathway receptors to augment specific response in cell culture and characterize transcriptomic/phenotypic response. This characterization will assist in NAMs effort to determine potential human health hazard of environmental chemicals of unknown effect.

Description:

Background and Purpose: The United States Environmental Protection Agency (US EPA) is heavily invested in developing higher-throughput, lower-cost New Approach Methodologies (NAMs) to identify potential hazards of thousands of environmental chemicals with unknown impacts to human health. Genetic editing techniques can mediate activation and inhibition of specific gene pathway targets of toxicological interest. Such manipulation can be used to generate molecular in vitro transcriptomic or phenotypic profiles that can aid in NAMs data interpretation for mechanism and potential hazards. In this study, we targeted specific activation of nuclear receptors of the retinoic acid (RA) pathway, a critical regulator of cellular development, differentiation, and proliferation, using a CRISPRa-dCas9 strategy in human osteosarcoma cells (U-2 OS). As reference chemical-mediated activation may have non-canonical or off-target effects, we compared RA pathway-mediated gene induction by CRIPSRa targeting with those altered by well-known retinoid receptor subtypes agonists. Methods: U-2 OS cells that stably express the CRISPRa-dCas9 system were transfected with a pool of targeting CRISPR RNAs (crRNAs) against either the alpha subtype of the retinoic acid receptor (RARA, using two different crRNA pools, p1 and p2) or the alpha or beta subtype of the retinoid X receptor (RXRA, RXRB). Transfection of a synthetic crRNA:tracrRNA pool complex was mediated by DharmaFECT 4 reagent using a 0.2µl:25nM reagent to complex ratio in each well. Control cells underwent the same process, but with a nontargeting crRNA. Transfection reagents were incubated with cells for 72 hrs before RNA isolation and target gene expression analysis with real-time quantitative PCR. RA pathway genes measured were retinoid receptors RARA, RARB, RXRA, and RXRB; the beta subtype of the retinoic acid metabolizer CYP26B, and reference genes GAPDH and HPRT1. Target gene expression was normalized to the geomean of GAPDH and HPRT1. Significant expression was determined by two-way t-test (p<0.05). Results: Our data demonstrated significantly increased expression of retinoic receptors RARA, RXRA and RXRB when activating RAR and RXR subtypes using CRISPRa-dCas9. When activating RARA (p1), we observed a 5-fold increase, a 48,000-fold increase, and a 4-fold increase in RARA, RXRA, and RXRB, respectively. When activating RXRA, we observed 5-fold, 72,000-fold, and 7-fold increase in RARA, RXRA, and RXRB, respectively. When activating RXRB, we saw 2-fold, 17-fold, and 3-fold increase in RARA, RXRA, and RXRB, respectively. The pool #2 (p2) of crRNA RARA was not as effective as p1; we observed a 1.5-fold change in RXRB and a 5-fold change in RARA, with no significant change in expression of RXRA (p>0.05). No conditions altered RARB or CYP26B1 expression significantly. Conclusions: We observed significantly increased gene expression of both retinoid acid or retinoic X receptor subtypes with crRNA-mediated activation of RARA, RXRA, or RXRB, presumably through auto-regulation (or direct gene expression increase mediated by the pooled crRNA). Unlike previously noted responses with RAR and RXR agonists exposure to U-2 OS cells, we did not observe increased expression of CYP26B1 and RARB with crRNA activation of receptors alone, likely due to lack of endogenous ligand. Given our goal is to refine transcriptomic and phenotypic profiles for RA pathway activation, we will modulate levels of retinoid receptor with crRNA with co-exposure to exogenous retinoid to determine canonical and non-canonical responses in future experiments. This case study will serve as a template for future studies of other toxicological gene pathway targets to develop and refine signatures of response that will help characterize the human health impact of environmental chemical exposure of unknown effect. This abstract does not necessarily reflect EPA policy. Mention of trade names is not an endorsement or recommendation for use.