215 -- Determining the Function of MHC Class I in Xenopus laevisUsing CRISPR/Cas9 Gene Editing

The MHC Class I protein plays a critical role in the immune system of Xenopus laevis. This protein functions in the determination between self and non-self. The MHC Class I protein is an important immune gatekeeper identifying foreign pathogens and ensuring that infected cells are killed by CD8 T cells. Also, it’s important to ensure that normal cells are not attacked by the immune system. In Xenopus laevis, it has been shown that MHC Class I molecules are found active in adult frogs, but have not been detected in tadpoles even though they are immunocompetent. We are interested to see if MHC Class I is critical for immune function in Xenopus tadpoles despite low to undetectable levels. By knocking out the MHC Class I gene in tadpoles we aim to determine its function. To do this, we utilized the CRISPR/Cas9 gene-editing tool. Cas9 creates a break in the dsDNA at the location of the gene by using specific guide RNAs, and the cell attempts to fix the break. This leads to mutations in the gene sequence that will inactivate the gene. We have generated multiple transgenic tadpoles using two sets of guide RNAs and are currently in the process of extracting DNA from both transgenic and control samples. We will use DNA sequencing to verify the successful knockout of the MHC Class I gene and will generate more transgenic animals to monitor the effects of gene inactivation on the phenotype of individual tadpoles.