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A method for CRISPR/Cas9 mutation of genes in fathead minnow
Citation:
Maki, J., T. Saari, A. Schroeder, J. Cavallin, G. Ankley, AND Dan Villeneuve. A method for CRISPR/Cas9 mutation of genes in fathead minnow. SETAC North America, Minneapolis, MN, November 12 - 16, 2017.
Impact/Purpose:
A method has been developed for CRISPR/Cas9 gene mutation in fathead minnow (Pimephales promelas), advancing our ability to assess gene function in this model organism. Now established, this system may be implemented to assess the function of any gene of interest, a situation particularly valuable for the validation and development of adverse outcome pathways (AOPs). This work will be continued in collaboration with The College of St. Scholastica, moving the guide strand development and genetic analysis into the classroom, exposing 10-20 upper level undergraduates to a research project each year. The microinjections and fish care will continue to occur at the EPA laboratories. This partnership will benefit the EPA by accelerating the pace of AOP validation through the classroom work in strand design and mutant characterization.
Description:
Product Description: CRISPR/Cas9 is a system that can be used to disrupt a gene of interest in any animal. It allows us to study each gene’s role by observing changes in the animal when the gene isn’t functional. We worked out a method to use this technology in the fathead minnow, a regionally important fish the EPA has studied for decades. The method provides many benefits; it not only allows us to gain a more complete understanding of the fish’s basic biology, it also reduces the number of fish used and the chemical exposure tests required. This means time and money are saved and fewer chemicals are consumed. Abstract: CRISPR/Cas9 genome editing allows for the disruption or modification of genes in limitless model organisms. Here, we discuss the method as developed for use with the fathead minnow (Pimephales promelas), in part to assist in validation of adverse outcome pathways (AOPs). Successful generation of CRISPR/Cas9 mutants in fathead minnow requires identification of a target gene, design of guide strands specific to the target, microinjection of Cas9/guide strand complexes into one cell embryos, growth and observation of phenotype, and finally genetic analysis of mutants. The tyrosinase gene 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 coding for the calcium binding domain 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 (IVF) was performed using a protocol developed for zebrafish (zfin.org). This allowed for microinjection of hundreds of developmentally synchronized embryos with Cas9 proteins complexed to each of the three guide strands. Fish were grown and observed for abnormal pigmentation and eventually anesthetized, photographed and flash frozen. DNA extraction of the larvae was followed by amplification of the potentially mutated region by PCR, and analysis of amplicons by sequencing or endonuclease assay to assess mismatches. 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. The contents of this abstract neither constitute nor necessarily reflect official US EPA policy.