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A method for CRISPR/Cas9 mutation of genes in fathead minnow (Pimephales promelas)
Citation:
Maki, J., J. Cavallin, K. Lott, T. Saari, G. Ankley, AND D. Villeneuve. A method for CRISPR/Cas9 mutation of genes in fathead minnow (Pimephales promelas). AQUATIC TOXICOLOGY. Elsevier Science Ltd, New York, NY, 222:12 pg., (2020). https://doi.org/10.1016/j.aquatox.2020.105464
Impact/Purpose:
CRISPR/Cas9 based gene editing is a recent technology that is having a profound impact on biological sciences. However, to date there has been only limited application of CRISPR/Cas9 directed gene mutation to understand the roles that specific biological targets or pathways play in mechanisms of chemical toxicity. The present manuscript reports on optimization and evaluation of methods to apply CRISPR/Cas9 approaches with the fathead minnow (Pimephales promelas), one of the most widely used species in aquatic toxicity testing. Mutation of a gene that regulates pigmentation in developing fish was successfully achieved, demonstrating the ability to adapt the technology for use with this important ecotoxicological model. Results of the case study inform the potential uses and current limitations for understanding adverse outcome pathways.
Description:
The fathead minnow, Pimephales promelas, is one of the most widely organisms for evaluating chemical hazards to the aquatic environment. Developing fundamental understanding of how chemicals cause toxicity in this organism, and related fish species, will aid the use of most cost effective and efficient alternatives to animal testing as a basis for chemical safety assessment. CRISPR/Cas9, a genome editing technology, has potential application for causing selective alterations to the genome that can significantly enhance our understanding of how chemicals cause toxicity. However, CRISPR/Cas9 had not been previously applied to this species. A number of technical hurdles to the application of CRISPR/Cas9 to this species were overcome and successful mutation of a gene regulating pigmentation in the fathead minnow was demonstrated. This study is an important step toward the routine use of CRISPR/Cas9 in aquatic toxicology research. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing allows for the disruption or modification of genes in a multitude of model organisms. In the present study, we discuss the method as developed for use with the fathead minnow (Pimephales promelas), in part, to assist in the development and validation of adverse outcome pathways (AOPs). The gene coding for an enzyme responsible for melanin production, tyrosinase, was the initial target chosen for development and assessment of the method since its disruption results in abnormal pigmentation, a phenotype obvious within 3 to 4 days after injection of fathead minnow embryos. Three tyrosinase-targeting guide strands were generated using the fathead minnow genome browser (setac.org/fhmgenome) in tandem with crispor.org. The strands targeted sequence in a conserved region that demonstrated homology to EGF-like or laminin-like domains as determined by Protein Basic Local Alignment Search Tool (BLASTp,) in concert with the Conserved Domain Database and the N-terminal region of the tyrosinase domain; both regions are found in the first half of the gene. In order to generate one cell embryos, in vitro fertilization was performed, allowing for microinjection of hundreds of developmentally-synchronized embryos with Cas9 proteins complexed to each of the three guide strands. Fish were observed for abnormal pigmentation after 3, 4, 14, and 34 days post-fertilization. DNA was extracted from larval whole bodies, amplified by PCR, and used to identify potential mutations by sequencing or endonuclease assays. This method greatly advances our ability to directly investigate gene function in fathead minnow, allowing for a more nuanced approach to AOP validation and development. It could also potentially produce novel molecular level insights into the basic biology of fathead minnow and other fishes.
URLs/Downloads:
DOI: A method for CRISPR/Cas9 mutation of genes in fathead minnow (Pimephales promelas)