Although gRNA-expressing lentiviral vectors efficiently modulate transcription, the cloning and the production of lentiviral particles will likely act as a technical bottleneck as this platform is expanded to higher-throughput applications. For one gene, using SNAP91 gRNA 3, we tested the efficacy of a transiently expressed in-vitro-transcribed (IVT) gRNA to modulate transcription in antibiotic-selected dCas9−VP64 NPCs. Forty-eight hours after transfection, we observed robust upregulation of SNAP91 in NPCs (C2, 6.13-fold, p < 0.001; n = 3 each; antibiotic selection for dCas9−VP64) (Figure 5A). This magnitude of response was comparable with that achieved by stable lentiviral transduction (Figure 2), suggesting that transient IVT gRNAs might represent a more scalable strategy moving forward. Efficacy of IVT gRNA-mediated transcriptional activation was confirmed across two additional neural genes, CEP162 (six pooled gRNAs) and FUT9 (ten pooled gRNAs) (Figure 5A). Finally, we compared the efficacy of pools of ten, five (two independent pools), and one IVT gRNA(s) to modulate transcription in antibiotic-selected dCas9−VP64 NPCs. Forty-eight hours after transfection, we observed greatest upregulation of FUT9 through multiplexing (C2, 10 gRNA pool, 3.3-fold, p < 0.05; n = 3 each; antibiotic selection for dCas9−VP64) (Figure 5B).
