CRISPR-CAS9 recombinant ADENO-ASSOCIATED VIRUS MEDIATED CYSTIC FIBROSIS GENE THERAPY (2023)
Undergraduate: Breanna Bowman
Faculty Advisor: Eric Hastie
Department: Biology
Cystic Fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. This research focuses on a 3-base pair deletion, F508d, which is a deletion of the amino acid phenylalanine. The F508d is the most common mutation among CF patients, with 90% possessing it. This study utilizes a CRISPR-Cas9 system and recombinant Adeno-Associated Virus (rAAV) to facilitate gene therapy for the F508 deletion. Using 5 different sgRNAs, 5 plasmid constructs were created and their correction efficiencies were tested.
A dual reporter, mRFP-508D-eGFP-CFTR, was created and used to test the homology-directed end repair efficiency of each sgRNA, 2-5, via human kidney cells, HEK293. The cells were then imaged using fluorescence microscopy to detect mRFP and eGFP. Fluorescing mRFP indicates successful transfection, while eGFP indicates a successful correction of the F508d. The cells were imaged with both RFP and GFP overlays to visualize which cells expressed both fluorescent proteins. The images were then analyzed to choose the most efficient sgRNA for this project. sgRNA5 showed the highest correction efficiency. Looking forward, the selected sgRNA, sgRNA5, will be transformed into rAAV-9 in order to be administered in mouse trials. These results show promise for potential rAAV-mediated gene therapy in human cystic fibrosis patients.