Chunk #8 — Results — The Efficacy of dCas9-Mediated Transcript Activation Varies Extensively between Genes and Is Not Necessarily Consistent between Unique Donors
A variety of human genes (VEGFA, NTF3 [Maeder et al., 2013], SOX2 [Kearns et al., 2014], and ASCL1, NEUROD1, MIAT1, and RHOFX2 [Chavez et al., 2015, Chavez et al., 2016]) have now been activated using dCas9 effectors in HEK293T cells or hiPSCs, although the extent to which any given gene is amenable to upregulation across a larger number of cell types is unclear; here we applied two well-established dCas9 transcriptional activators, dCas9−VP64 and dCas9−VPR, to hiPSC-derived NPCs and neurons (Figures 1A and 1B). As extensive transcriptional variability exists between hiPSCs from different donors (Carcamo-Orive et al., 2017, Nishizawa et al., 2016, Rouhani et al., 2014), we first considered how well the activating capabilities of both platforms translated across five genes (Figure 1C) using neural cells from three unique donors. For each gene, gRNAs were designed to target at least three distinct locations upstream of the transcriptional start site (TSS), and thus within the putative promoter elements (Figure 1C; Table S2). Both antibiotic-selected (Figure S2) and non-selected lentiviral-transduced dCas9−VP64 and dCas9−VPR NPCs were evaluated.
