Chunk #10 — Results — The Efficacy of dCas9-Mediated Transcript Activation Varies Extensively between Genes and Is Not Necessarily Consistent between Unique Donors
Second, across six gRNAs generated to the promoter regions to NRXN1, we failed to observe robust increases in expression with either dCas9−VP64 or dCas9−VPR NPCs (Figures 2C and S3), only achieving increased NRXN1 expression in one individual with one gRNA using either the dCas9−VP64 and dCas9−VPR effectors (gRNA 2: dCas9−VP64 C3, 5.66-fold, p ≤ 0.0001; dCas9−VPR C3, 8.53-fold, p ≤ 0.05; n = 3 each; antibiotic selection for dCas9−VP64 and dCas9−VPR) (Figure 2C). Three additional gRNAs, designed upstream of a second TSS, were also unable to increase NRXN1 expression, with or without selection for dCas9−VP64 in one individual (Figure S3), demonstrating that testing up to six gRNAs does not ensure successful gene activation.
