OPTIMIZATION OF GENE DELIVERY SYSTEMS FOR EFFICIENT GENOME EDITING APPLICATIONS

Abstract

This paper will establish an efficient system of gene delivery to reduce cytotoxicity and off-target effects at the maximum level of efficiency of gene/genome editing. Four delivery systems namely adeno-associated viral vectors (AAV), lipid nanoparticle (LNP), polymeric nanoparticle (PEI-based) and electroporation with CRISPR-Cas9 editing activity in HEK293 and HeLa cells were compared and evaluated. The rate of transfection, genome editing and cytoskeleton viability and off-target were quantitatively measured with flow cytometry and T7E1 assay, as well as sequencing-based analysis. These findings showed that the greatest transfection efficiency of 86.3 % with a standard deviation of 2.8 was observed with LNP-based delivery, then electroporation (78.5 % with a standard deviation of 3.1 %), AAV (72.4 per cent with a standard deviation of 2.5 %) and polymeric nanoparticles (64.7 per cent with a standard deviation of 3.6 %). The maximum efficiency of genome editing was obtained with optimised LNP systems with 81.2% reporting of the efficiency with a variation of 2.4%. This is 27% higher than the efficiency of a delivery using a polymer. The highest viability of the cells treated with LNP (91.6% 2.2) was observed, in comparison with electroporation (74.3% 3.8) revealing less cytotoxicity. Moreover, the frequency of off-target mutation was also much less in the rib nucleoprotein (RNP)-based delivery (3.1 per cent) than in plasmid-based systems (8.7 per cent) (p < 0.01). The results show that optimised lipid nanoparticle-mediated delivery is an excellent compromise of efficiency, precision, and biocompatibility, thus showing promise in scalable genome editing and therapeutic gene deliveries using lipid nanoparticles as a delivery method.