gRNA positional efficacy for activation (Figure 2) and repression (Figure 3) was somewhat consistent with SNAP91 (gRNA 2 > gRNA 3/1) and CLCN3 (gRNA 3 > gRNA 1/2). Similar to results with dCas9−VPR, we found that SNAP91 gRNA 2 had the most profound repressing effect in day 8 NGN2 neuron populations (gRNA 2: C1, 0.43-fold, p < 0.0001; C2, 0.31-fold, p < 0.0001; C3, 0.24-fold, p < 0.0001; n = 3 each; no antibiotic selection for dCas9−KRAB) (Figure 3C). SNAP91 gRNA 2 also robustly decreased SNAP91 levels in day 20 NGN2 neurons (C1, 0.77-fold, p < 0.001; n = 9 each; antibiotic selection for dCas9−KRAB) (Figure 3D). Surprisingly, when these same three gRNAs were tested in NPCs (rather than NGN2 neurons) from these same three individuals, gRNA 3 rather than gRNA 2, showed greatest efficacy (C1, gRNA 2; 0.45-fold, p < 0.0001/gRNA 3; 0.29-fold, p < 0.0001; C2, gRNA 2; 0.46-fold, p < 0.0001/gRNA 3; 0.28-fold, p < 0.0001; C3, gRNA 2; 0.33-fold, p < 0.0001/gRNA 3; 0.19-fold, p < 0.0001; n = 3 each; antibiotic selection for dCas9−KRAB) (Figure
