Author: Madalyn Wheeler
Approved: Spring 2020
The goal of this study is to use CRISPR/Cas9 to induce a targeted mutation in the StAR gene, which encodes a protein that is mutated in the human disease Lipoid Congenital Adrenal Hyperplasia (LCAH). The StAR protein is crucial for the biosynthesis of all steroid hormones from cholesterol, including estrogen and testosterone. The zebrafish star gene is nearly identical to the human StAR gene, and a frameshift mutation in this gene should induce developmental defects similar to those that occur in human LCAH. Thus far, Cas9 DNA has been cloned from an E. coli plasmid and transcribed to generate Cas9 mRNA. An additional two guide DNA constructs were cloned, transcribed, and the presence of gRNA was confirmed by nanodrop assay. Gel electrophoresis will be used to screen CRISPR- injected embryos for the predicted 40 bp deletion between cut sites. Once injections of the Cas9/gRNA solution begin embryos will be analyzed by gel electrophoresis to see if a deletion has occurred. When there appears to be a successful injection there will be further analysis of injected embryos to confirm a mutation. After confirmation, injected fish will be raised to adulthood. Mosaic mutants will be cross-bred with wild-type fish to create a stable, heterozygous mutant line. Once a heterozygous star mutant line is established, further research will be done on the homozygous star mutant progeny. These homozygous mutant embryos should exhibit both the physiological and biochemical symptoms of human Lipoid Congenital Adrenal Hyperplasia.