Creation of a new speedbump plasmid and allele in zebrafish using CRISPR-Cas9
Kelly, Jackson C.
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Zebrafish are commonly used as a model organism to study developmental genetics and the cell cycle. They are vertebrates that develop very quickly, and therefore their development and anything that influences it can have great research implications. The speedbump mutant is one of the early arrest mutants of the Zebrafish. This phenotype causes embryonic death within 18 hours of fertilization. The phenotype has irregular growth and dark cells particularly around the notochord of the embryo. This mutation is believed to be caused by the wee1 gene. Using a Caspase-3 antibody stain, the darkened cells near the notochord were shown to be apoptotic. Embryos were also analyzed with BRDU and DAPI staining with inconclusive results. A CRISPR-Cas9 construct was made for the wee1 zebrafish gene, which will be used to try to rescue a speedbump fish into a wildtype fish. This construct was sequenced and shown to be made properly using the CRISPR-Cas9 cleavage assay. If the phenotype can be rescued using the construct, the phenotype is proven to be caused by the wee1 gene. This phenotype and these findings can be used for the potential reversal of the apoptotic process, which could have therapeutic applications for cancer and other debilitating diseases.