168 STING-IFN-κ-APOBEC3G pathway mediates resistance to CRISPR transfection in keratinocytes

CRISPR-Cas9 has been proposed for treatment of genetically inherited disorders including of the skin. However, a limitation for widespread use of CRISPR for correction of inherited skin diseases is the poorly understood transfection resistance of keratinocytes (KCs). Here we report that CRISPR transfection activates STING dependent antiviral responses in KCs, resulting in heightened endogenous interferon (IFN) responses (p < 0.01) through induction of IFNκ (p < 0.001), and decreased plasmid stability secondary to induction of the cytidine deaminase APOBEC3G. Notably, CRISPR generated KO KCs had permanent suppression of IFNκ (p < 0.001) and IFN stimulated genes (ISGs) expression (p < 0.001), secondary to hypermethylation of the IFNK promoter region by the DNA methyltransferase DNMT3B. Pre-treatment with the JAK1/JAK2 inhibitor, baricitinib prior to CRISPR transfection led to enhanced transfection efficiency (p < 0.001), absence of IFNK promoter hypermethylation, and normal IFNκ activity and ISG responses. These results provide insights into the transfection resistance of KCs and indicate that CRISPR mediated gene-correction can lead to permanent alteration of antiviral responses in skin, which can be prevented by JAK1/JAK2 inhibition. This work has major implications for future gene therapy of inherited skin diseases using CRISPR technology.