Given that chromatin states, particularly nucleosome positioning, can differ between cell types (Jiang and Pugh, 2009) and that nucleosomes can impede Cas9 access to DNA (Horlbeck et al., 2016b, Isaac et al., 2016), we next set out to determine whether gRNA activity in NPCs or neurons is predictive of efficacy in astrocytes. We differentiated astrocytes (NPC-astrocytes) (TCW et al., 2017, Xu et al., 2016) (Figure 4A) from the antibiotic-selected dCas9-VP64 NPC lines. NPC-astrocytes are positive for the astrocyte markers S100β, GFAP, and EAAT1 (Figures 4A and 4B), and up to 90% of antibiotic-selected NPC-astrocytes were positive for dCas9 protein by fluorescence-activated cell sorting (FACS) (Figure 4C).
