Development of gene editing strategies for human β-globin (HBB) gene mutations

Recent developments in gene editing technology have enabled scientists to modify DNA sequence by using engineered endonucleases. These gene editing tools are promising candidates for clinical applications, especially for treatment of inherited disorders like sickle cell disease (SCD). SCD is caused by a point mutation in human β-globin gene (HBB). Clinical strategies have demonstrated substantial success, however there is not any permanent cure for SCD available in clinics. CRISPR/Cas9 platform uses a single endonuclease and a single guide RNA (sgRNA) to induce sequence-specific DNA double strand break (DSB). In this study, it was aimed to target HBB gene via CRISPR/Cas9 genome editing tool to introduce nucleotide alterations for efficient genome editing and correction of point mutations causing SCD in HEK293T cell line, by Homology Directed Repair (HDR). We have achieved to induce target specific nucleotide changes on HBB gene in the locus of mutation causing SCD. The effect on on-target activity of longer sgRNA was examined and observed that longer sgRNA has the same performance for targeting and Cas9 induced DSBs. HDR mechanism was triggered by co-delivery of donor DNA repair templates in circular plasmid form. In conclusion, we have suggested methodological pipeline for efficient targeting and creating desired modifications on HBB gene