Methylmercury (MeHg) is a common water pollutant in the Great Lakes region with known toxicity in wildlife and humans. The glutathione (GSH) pathway plays a crucial role in detoxifying cells from substances like MeHg. While variants in GSH genotypes have been linked to differential MeHg retention and toxicity in adult tissues, less is known about these gene-environment interactions during embryonic development. To investigate this context, a mutant line of zebrafish was created utilizing CRISPR techniques targeting the gclm gene, a gene involved in the GSH pathway. In this study, we characterized the mutant allele in the F3 generation using high-resolution melting analysis (HRMA) along with PCR + restriction enzyme digestion. Suspected mutant zebrafish were genotyped to detect a change in DNA sequence at the CRISPR target area using PCR + restriction enzyme digestion with gel electrophoresis. Gel extraction of the altered PCR fragment and subsequent DNA sequencing revealed a consistent deletion at the CRISPR target area. Subsequent analysis of predicted protein products of the mutant sequences indicated a loss-of-function allele. Characterizing the functional impact of this mutant gclm allele will help clarify how the gclm gene contributes to MeHg detoxification in the context of embryonic development.
