Transcriptional activation in dCas9−VP64 NPC-astrocytes (Figures 4D and 4E) was inconsistent with results observed in dCas9−VP64 NPCs and neurons (Figures 2A, 2D, and 2E). For KCTD13, only gRNA 2, and only in one individual, increased expression (C2, 1.28-fold, p < 0.05; n = 3; antibiotic selection for dCas9−VP64). Unexpectedly, NPC-astrocytes were surprisingly more amenable to transcriptional activation of SNAP91 (Figure 4E) than NPCs or neurons (Figures 2D, 2E, and 2F). SNAP91 gRNAs 2 and 3 produced dramatic increases in expression in NPC-astrocytes, two orders of magnitude larger than those observed in NPC or neurons (C1, gRNA 2; 458.5-fold, p < 0.0001/gRNA 3; 405.5-fold, p < 0.0001; C2, gRNA 2; 248.4-fold, p < 0.0001/gRNA 3; 198.4-fold, p < 0.0001; C3, gRNA 2; 564.7-fold, p < 0.0001/gRNA 3; 467.7-fold, p < 0.0001; n = 3 each; antibiotic selection for dCas9−VP64) (Figure 4E). Multiplexing of SNAP91 gRNAs 1, 2, and 3 achieved greater efficacy than any single gRNA alone (Figure 4E).
