The enhancement of CRISPR/Cas9 gene editing using metformin

Abstract

The CRISPR/Cas9 technology is a revolutionary tool that can be used to edit the genome. Specifically, the genome of hematopoietic stem cells (HSCs) could be edited to correct monogenic blood disorders as well as produce immunotherapies. However, the efficiency of editing HSCs remains low. To overcome this hurdle, we set out to investigate the use of metformin, an FDA-approved drug, to enhance gene modification. We assessed the effect of metformin on the growth of two hematopoietic cell lines: a myeloid-erythroid leukemic cell line (K562 cells) representative of the myeloid population and an immortalized T lymphocyte cell line (Jurkat cells) representative of the lymphoid population. No significant difference in growth patterns was observed in concentrations up to 10 mM metformin in both cell lines. We then assessed the ability of two different concentrations of metformin (0.001 mM or 1 mM), based on our observations, to enhance both (1) the cutting efficiency of Cas9 and (2) the targeting efficiency with the use of a donor DNA repair template. The cutting efficiency of Cas9 was significantly enhanced in a total of five guide RNAs (four specific to a platelet locus and one specific to an erythroid locus) following treatment. In addition, an enhancement in targeting was observed with the use of a GFP-containing donor DNA repair template with both concentrations. Overall, a greater than two-fold increase in GFP expression was noted in cells treated with metformin. This suggests that metformin, an FDA-approved drug, could be added to existing protocols to enhance CRISPR/Cas9 gene editing.

Keywords

• CRISPR
• Gene editing
• Metformin