Therapeutic genome editing using the CRISPR/Cas9 system

Abstract

Disease X is one of the most common secondary complications of diabetes mellitus (DM) worldwide. Studies have showed that Gene Y (the first enzyme of pathway Z) plays an important role of the development of Disease X under hyperglycemia. Disease X has not only negative impact on human life but also has a significant economic burden. CRISPR (Clustered, Regularly Interspaced, Palindromic Repeats) is the latest advanced gene editing technique. This gene editing system offers a significant potential for prevention and treatment of diseases caused by genetic disorders. This master’s project aims to investigate if knockout of Gene Y by CRISPR/Cas9 system could have a therapeutical and protective effect against Disease X. In this study, in vitro transfection parameters (gene delivery, transfection reagent selection, guide plasmid selection, experimental steps etc.) were first optimized to provide efficient transfection parameters for future in vivo transfection. Gene deletion was first verified by PCR-based screening. Sanger sequencing was also done to verify that guide RNA sequences were successfully introduced to target the mutation sites of host genome. In summary, final transfection was performed based on these optimal conditions and reagents, the Cas9 and two guide RNAs were introduced non-virally to determine if the knockout of the Gene Y in these cells could have an impact on Protein Y expression level and activity. The efficiency of knockout was determined by measuring the reduction of Protein Y by Western blot.