There was surprisingly little correlation between gRNA efficacy in neural cells and HEK293T cells. While KCTD13 gRNAs 2 and 3 proved efficacious in some (but not all) control dCas9VP64 NPCs, it was gRNAs 1 and 3 that increased expression in antibiotic-selected, but not unselected, dCas9−VP64 HEK293Ts (Figure S5A). Although TOAK2 gRNAs failed to increase expression in dCas9VP64 NPCs, gRNA 2 increased expression in antibiotic-selected, but not unselected, dCas9−VP64 HEK293Ts (Figure S5B). Unexpectedly, NRXN1 gRNAs 1–6 somewhat increased expression in antibiotic-selected dCas9−VP64 HEK293Ts, but not antibiotic-selected dCas9−VPR HEK293Ts (Figures S5C and S5D). Even more unexpectedly, while validated CLCN3 gRNA 2 increased expression in dCas9−VP64 HEK293Ts (Figure S5E), validated SNAP91 gRNA 3 failed to increase expression in dCas9−VP64 HEK293Ts under any conditions (Figure S5F).
