GENOTYPIC RESULT OF THE INSULIN-LIKE RECEPTOR GENE KNOCKOUT IN DAPHNIA PULEX EB1

Abstract

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 has been used in multiple gene modification studies in various organisms. Daphnia, water fleas, are used in ecological studies due to their ability to sense and respond to environmental factors. Their quick life cycles make them prime candidates for gene modification and evolutionary studies. In our experiment, Daphnia pulex EB1 was used in the knockout of the Insulin-like Receptor gene via CRISPR-Cas9 due to its effect on dwarfism in humans and developmental disorders in mice. Polymerase Chain Reaction (PCR) sequencing was done to compare the EB1 gene of interest to the PA42 reference genome, generating a single guide RNA (sgRNA). There were occasional single nucleotide polymorphisms (SNPs); to overcome this, the sgRNA was designed to match the EB1 sequence. In-vitro testing and gel electrophoresis was performed on the digested sample to confirm the knockout was successful. In-vivo studies with PCR will need to be performed in the future to further the advancement of CRISPR-Cas9 studies.