Targeted activation of the human IL1RN and HBG1/2 genes by TALE-VP64 and dCas9-VP64 transcription factors. (A) Four TALEs (blue) and four gRNAs (orange), each labeled A through D, were designed to target the IL1RN and HBG1/2 promoters within the ∼200 bp upstream of the transcriptional start site (TSS; green). The position of each TALE and gRNA is shown to scale. (B) Single expression plasmids or combinations of two, three, or four expression plasmids for the TALE-VP64s or gRNAs, along with dCas9-VP64, targeted to each gene were transfected into HEK293T cells. Expression of the target gene was assessed by qRT-PCR. Robust gene activation was observed only in response to the combination of TALE-VP64s or gRNAs with dCas9-VP64. (C) The VP64 activation domain is essential for target gene induction. HEK293T cells were transfected with the combination of expression plasmids for the four TALEs with and without the VP64 domain, or four gRNAs either alone or with dCas9 or dCas9-VP64. Only samples transfected with TALE-VP64s or gRNAs with dCas9-VP64 showed changes in target gene expression. Gene expression is normalized to GAPDH levels and shown as fold-increase relative to control cells transfected with an empty expression plasmid (mean ± SEM, n = 4 independent transfections across two experiments, different letters indicate P < 0.0001 by Tukey's test after log transformation).
