122 - Using CRISPR/Cas9 Genome Editing to Knockout MHC Class I in Xenopus laevis

The immune system of the frog Xenopus laevis is similar to humans. MHC Class I is a vital molecule for the immune system of Xenopus laevis. It presents peptides to CD8 T-cells and the presentation of self peptide fragments is crucial for immune self recognition. When MHC Class I presents non-self peptide fragments, it triggers an immune response, causing CD8 T-cells to kill the infected cells. All cells express some level of MHC Class I because all cells can be infected. The role this molecule plays in immune function and self recognition is of particular interest in Xenopus laevis since tadpoles are immunocompetent, yet have undetectable levels of MHC Class I protein (mRNA can be detected in different tissues). MHC Class I protein levels become detectable after metamorphosis and are expressed both as mRNA and protein in adult frogs. We are interested to see if MHC Class I is critical for immune function in Xenopus laevis tadpoles and will investigate by knocking out the MHC Class I gene. 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 while the cell attempts to fix its genome multiple insertions and/or deletions can occur in the sequence that inactivates the gene. We generated transgenic tadpoles that have guide RNAs targeting the MHC class I gene. Currently, we are using DNA sequencing to verify successful knockout of the MHC Class I gene

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