CRISPR-Cas9-Mutated Pregnane X Receptor (pxr) Retains Pregnenolone-induced Expression of cyp3a65 in Zebrafish (Danio rerio) Larvae

Abstract

Abstract Pregnane X receptor (PXR; NR1I2) is a nuclear receptor that regulates transcriptional responses to drug or xenobiotic exposure, including induction of CYP3A transcription, in many vertebrate species. PXR is activated by a wide range of ligands that differ across species, making functional studies on its role in the chemical defensome most relevant when approached in a species-specific manner. Knockout studies in mammals have shown a requirement for PXR in ligand-dependent activation of CYP3A expression or reporter gene activity. Morpholino knockdown of Pxr in zebrafish indicated a similar requirement. Here, we report on the generation of 2 zebrafish lines each carrying a heritable deletion in the pxr coding region, predicted to result in loss of a functional gene product. To our surprise, larvae homozygous for either of the pxr mutant alleles retain their ability to induce cyp3a65 mRNA expression following exposure to the established zebrafish Pxr ligand, pregnenolone. Thus, zebrafish carrying pxr alleles with deletions in either the DNA binding or the ligand-binding domains did not yield a loss-of-function phenotype, suggesting that a compensatory mechanism is responsible for cyp3a65 induction. Alternative possibilities are that Pxr is not required for the induction of selected genes, or that truncated yet functional mutant Pxr is sufficient for the downstream transcriptional effects. It is crucial that we develop a better understanding for the role of Pxr in this important biomedical test species. This study highlights the potential for compensatory mechanisms to avoid deleterious effects arising from gene mutations.