Optimization of CRISPR-Cas9 Construct to Correct Cystic Fibrosis Mutation F508del-CFTR

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, and 90% of CF patients possess the mutation F508del-CFTR. One potential treatment for CF patients is recombinant adeno-associated virus (rAAV) gene therapy using CRISPR-Cas9 gene-editing technology. This study focused on the design and optimization of an rAAV-CRISPR-Cas9 cassette to correct F508del-mCFTR. Five CRISPR-Cas9 constructs were constructed with different sgRNA sequences (1-5). Correction efficiency of the CRISPR-Cas9 construct with sgRNA3 was tested with a dual reporter mRFP-mCFTRF508del-eGFP plasmid in HEK 293 cells. mRFP and eGFP were detected via fluorescence microscopy two days after transfection. mRFP fluorescence indicated transfection, and eGFP fluorescence indicated successful CRISPR-Cas9 editing of F508del-mCFTR. The sgRNA3 CRISPR-Cas9 construct resulted in F508del-mCFTR gene editing, as indicated by eGFP expression in combination with mRFP expression. Future research includes optimization of transfection of the dual reporter system and CRISPR-Cas9 constructs, testing of other sgRNA CRISPR-Cas9 constructs, and development of an rAAV-CRISPR-Cas9 cassette using the most efficient sgRNA CRISPR-Cas9 construct. This cassette will be tested in F508del-CFTR mouse models and has the potential to inform human gene therapy approaches.Bachelor of Scienc