352 Allelic series for an EDC enhancer generated by CRISPR/Cas9 genome editing identifies an enhancer requirement for proximal gene expression and efficient wound healing

Abstract

The epidermal-specific 923 enhancer is involved in dynamic chromatin remodelling and activation of the Epidermal Differentiation Complex (EDC) locus. To test the function of 923 during epidermal development, we used the CRISPR/Cas9 genome editing system to generate multiple 923 genetic alleles using 2 guide RNAs and 2 loxP ssODNs targeted to the mouse 923 locus. Germline transmission of 2 independent 923 global deletions [923dela, 923delb], one 923 inversion [923inv], and one floxed 923 was confirmed with at least 4 rounds of C57BL/6 backcrosses to exclude off-target effects for each allele. 923inv enabled the study of the effect of enhancer directionality on developmental EDC activation. 923dela-/-, 923delb-/-, and 923inv-/- mice are fertile, viable, and appear normal. No significant changes in the EDC gene expression of newborn 923inv-/- skin were observed, consistent with the direction-independent function of classical enhancers. However, a dose-dependent decrease in the expression of the EDC genes proximal to 923 (involucrin (Ivl), Sprr3, and Sprr2d) was identified in the heterozygous and homozygous mice of the 2 independent 923 deletions (923dela, 923delb). Coincident with this finding and contrary to previous studies of Ivl KO mice, we also observed a dose-dependent and concomitant upregulation of loricrin and Flg-like gene family members (filaggrin, filaggrin-2, hornerin, and repetin) in the EDC, as well as envoplakin (Evpl) and periplakin (Ppl) that, with Ivl, form the cornified envelope (CE) scaffold. Delayed wound closure in both 923del-/- lines (F2) was also noted. Thus, we find that 923 is required for proximal gene expression (Ivl, Sprr3, Sprr2d) and recovery of the skin barrier after perturbation, and elucidate a distinct molecular signature for upregulation of the Flg-like gene family and CE scaffold components to maintain barrier integrity in the context of loss of 923 enhancer and downstream Ivl expression.