Fig. 2

Establishment of the enChIP method. A Graphical summary of the enChIP approach. B Induction of MICA gene expression through the SP-dCas9-VPR/gRNA activation system. RT-qPCR was performed 48 h after transient transfection of HEK293 cells with dCas9/VPR (Rta, p65, VP64) and sgRNAs 1–8 targeting the MICA promoter locus, a mix of sgRNAs 1,2,4,6, and 8, as well as a HBG promoter locus-targeting sgRNA as a positive control and a GFP-targeting one as a negative control. PAM: protospacer adjacent motif, TSS: transcription start site. The y-axis for HBG depicts Cy0 for the cycle threshold (CT) at which a measurement signal was present during cyclic amplification. C enChIP using the pEF1a-FB-dCas9/gRNA system to isolate the MICA promoter genomic region. Transient transfections of HEK293 cells with pEF1a-FB-dCas9 and a mix of sgRNAs to target the MICA promoter locus, as well as GFP-targeting control sgRNA were performed. Transfected cells were subjected to enChIP and the target locus was isolated using dCas9-targeting immunoprecipitation. RT-qPCR analysis was performed to validate an enrichment of the MICA promoter, for which the PDK4 region was used as a negative control. Data (B, C) represent the mean (±SEM) of three or four biological replicates. Statistical analysis was performed with an unpaired student's t-test