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Use of CRISPR/Cas9-mediated gene disruption in Xenopus tropicalis to evaluate thyroid-related targets of toxicological concern
Citation:
Haselman, J., J. Christensen, J. Olker, Pat Kosian, K. Donnay, P. DeGoey, M. Hornung, AND S. Degitz. Use of CRISPR/Cas9-mediated gene disruption in Xenopus tropicalis to evaluate thyroid-related targets of toxicological concern. SETAC North America, Sacramento, CA, November 04 - 08, 2018.
Impact/Purpose:
This work demonstrates the utility of CRISPR/Cas9 gene editing for quantitative AOP characterization in lieu of chemical exposures. This technique has the potential to significantly reduce animal usage for toxicity testing with the ability to elicit highly specific responses, which supports AOP discovery and development and will advance EPA toward a predictive toxicology framework that will replace animal testing for chemical safety evaluation.
Description:
CRISPR/Cas9-mediated gene editing is now an easily employed straightforward tool for studying gene function and has logical applications in the field of toxicology. Functional knockout of a protein implicated as a toxicological target serves as a highly specific method to evaluate downstream biochemical and apical effects when a molecular target is perturbed - in lieu of model chemical exposures. This approach could prove most useful for molecular targets that are not yet well-characterized by traditional toxicological approaches involving exposures to model chemicals or for targets where model chemicals are unidentified. To demonstrate the effectiveness of gene editing for thyroid disruption research, we employed the CRISPR/Cas9 system to knock out the thyroperoxidase (TPO) gene in model amphibian species Xenopus tropicalis. Newly fertilized embryos were microinjected with CRISPR/Cas9 ribonucleoprotein (RNP) complexes designed to target three different sites within the TPO gene. The viable larvae for each of the RNP-injected, non-injected and mock-injected groups were grown out in flow-through tanks for continued evaluation. All of the surviving non-injected controls and mock-injected larvae completed metamorphosis with a median metamorphosis time of 48 days (n = 85, 42, respectively). Of the surviving RNP-injected larvae, 70 out of 89 completed metamorphosis with a median metamorphosis time of 48 days while 19 putative knockouts were arrested at Nieuwkoop and Faber stage 55 (onset of pro-metamorphosis). This arrested development phenotype was consistent with previous studies of TPO inhibition by exposure to potent model TPO inhibitors. The DNA of larvae with putative TPO knockout was confirmed to have persistent mutations in the targeted regions of the TPO gene by Sanger sequencing, supporting the specificity of the apical effects. As demonstrated here with a well-characterized mechanism of thyroid toxicity, CRISPR/Cas9-mediated gene editing will support the linkages between a putative molecular target, downstream biochemical effects and adverse organismal outcomes, facilitating the advancement of predictive approaches to chemical safety evaluation.