Methods of Mutation Efficiency Analysis for CRISPR/Cas9 in Fathead Minnow

CRISPR/Cas9 mutation of fathead minnow genes allows for assessment of the key event relationships highlighted in Adverse Outcome Pathways (AOPs), diagrammatic representations of biological data from chemical exposure experiments. An AOP developed to hypothesize how mercaptobenzothiazole (MBT) exposure results in decreased anterior swim bladder inflation in fathead minnow reasons that MBT inhibits thyroid peroxidase (TPO), leading to a decrease in T4 hormone synthesis and the swim bladder phenotype. In vitro fertilization and microinjection methods for fathead minnow were previously developed in collaboration with the Environmental Protection Agency research labs, and verified by successful CRISPR/Cas9 tyrosinase mutation. Tyrosinase mutant fish were identified by altered pigmentation and genotypically confirmed by Sanger sequencing and TIDE analysis (Tracking of Indels by Decomposition; tide.nki.nl). In a continued collaborative effort, mutation of TPO and additional AOP‐related targets have been attempted and assessment of possible mutant fish is ongoing. In this work, methods of mutation analysis were assessed, including T7 endonuclease I (T7EI) kit, TIDE analysis and assessment via DNA Bioanalyzer. Results of the T7EI protocol with TPO experimental fish provided evidence of mutations, however, both the TIDE and Bioanalyzer data showed that mutation efficiencies were low to nonexistent in these fish. This is consistent with the lack of observable phenotype. Since the CRISPR/Cas9 attempt was not successful in generating mutations, the validity of the TPO AOP can neither be supported nor refuted until guide strand design is optimized to induce a higher mutation efficiency. Current efforts are focused on guide strand redesign for a second attempt at TPO mutation. Additional targets are simultaneously being pursued for mutation with the in vitro fertilization and CRISPR/Cas9 system. Based on the data obtained here, TIDE analysis will be the primary technique utilized for assessment of mutation efficiency in subsequent studies. Though time‐consuming, TIDE analysis has proven reliable, provides clear results and is cost‐effective. Support or Funding Information U.S. Environmental Protection Agency, Office of Research and Development, Mid‐Continent Ecology Division, Duluth, MN. The College of St. Scholastica Faculty Development Committee Research Grant. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .