Chunk #11 — Results — The Efficacy of dCas9-Mediated Transcript Activation Varies Extensively between Genes and Is Not Necessarily Consistent between Unique Donors
Third, highly expressed in neurons (Zhang et al., 2016), SNAP91 and CLCN3 transcriptional modulation was tested in NEUROGENIN2 (NGN2)-induced populations of excitatory neurons (Ho et al., 2016, Zhang et al., 2013) after 8 days of maturation. Following transduction of three distinct gRNAs to SNAP91, in parallel with a lentivirus containing inducible NGN2, we found that gRNA 2 significantly increased SNAP91 levels in dCas9−VPR day 8 NGN2 neurons from all three donors (gRNA 2: C1, 1.33-fold, p ≤ 0.01; C2, 1.66-fold, p ≤ 0.01; C3, 2.02-fold, p ≤ 0.001; n = 3 each; no antibiotic selection for dCas9−VPR) (Figure 2D), results that were confirmed by western blot for gRNA 2 (n = 1, antibiotic selection for dCas9−VPR) (Figure S4A). SNAP91 gRNA 2 also produced robust increases in SNAP91 levels in NPCs (C1, 16.31-fold, p < 0.0001; n = 9 each; antibiotic selection for dCas9−VPR) (Figure 2E). Of the three CLCN3 gRNAs evaluated, only gRNA 3 increased expression in dCas9−VPR NPCs (C2, 1.20-fold, p ≤ 0.05; C3, 1.27-fold, p ≤ 0.01; n = 3 each; no antibiotic selection for dCas9−VPR) (Figure 2F).
