Plasmid Construction and Cloning of dCas9-p300 Fusion and gRNAs

Plasmids encoding active and mutant forms of a deactivated-Cas9-histone acetyltransferase P300 fusion protein (pcDNA-dCas9-p300 Core, pcDNA-dCas9-p300 Core-D1399Y) and guide RNA only expression vector (phU6-gRNA) were gifts from Charles Gersbach (Hilton et al., 2015 (link)) (Addgene #61357, #61358, and #53188, respectively). DER-specific gRNAs were designed by submitting DER coordinates or sequence to the CRISPOR¹ and Breaking-Cas (Oliveros et al., 2016 (link)) servers, respectively, and selected based on overlap with BEC DNAse hypersensitivity sites (ENCODE). Control gRNAs to IL1RN were described previously (Perez-Pinera et al., 2013 (link)). gRNAs were annealed and cloned into phU6-gRNA via BbsI restriction sites (NEB #R0539S). All plasmids were transformed into OneShot^®TOP10 competent cells (Thermo Fisher Scientific), cultured overnight, and extracted using Endo Free^® Plasmid Maxi or QIAprep^® Spin Miniprep (gRNAs) kits (QIAGEN). gRNAs sequence and locations are listed in Supplementary Table S5.