386 Autocrine IFN-k restricts CRISPR-Cas9 keratinocyte transfection

Abstract

CRISPR-Cas9 is a unique genome-editing tool but is limited in regards to variable transfection efficiency particularly in keratinocytes (KCs) (1-2%), in contrast to HEK293T cells (62%). Despite this, we have successfully generated >20 knockout lines (KO) in immortalized KCs. Intriguingly, in all KO generated, IFNK mRNA expression (p= 1.7E-06), an endogenous type I IFN secreted by KCs, as well as expression of IFN response genes (IRF7, p<0.01; MX1, FC, p<0.01) were significantly suppressed in all KO lines. The decreased IFNK mRNA expression correlated with hypermethylation of the promoter region in KO lines. Notably, KO KCs (for any gene tested) had almost a 10-fold (p<0.01) increase in transfection efficiency compared to control KCs. In contrast, we observed marked suppression (FC=0.1, p<0.01) of transfection efficiency when recombinant type I IFN was added to cell culture medium prior to transfection. CRISPR plasmid uptake by KCs was highest at 6% at 12 hrs and decreased at later time point (24hrs (3%), 48 hrs (1%)), and accompanied by increased IFNK and MX1 mRNA expression (FC=3.74, p<0.001 and FC=10.72, p<0.001 respectively) at 24 hr time point. This IFN response was dependent upon the function of the intracellular RNA sensor STING. Our data suggest that CRISPR activates IFN-kappa through STING-dependent pathways and that activation of endogenous KC type I IFN signaling is responsible for KC resistance to CRISPR transfection. These findings will have major implications regarding CRISPR research and its use to correct epithelial defects.