Overall, these results demonstrate that gRNAs designed upstream of the TSS and validated for dCas9−VP64 or dCas9−VPR should not be assumed to be effective when combined with dCas9−KRAB. As observed with gene activation, dCas9−KRAB results are not always consistent across unique individuals. Again, this variation could not be explained by differences in the levels of dCas9-effector protein (Figure S2A) or transcript (Figures S2B and S2C) between individuals. Surprisingly, in at least one case, gRNA efficacy in NPCs did not predict efficacy in neurons. We recommend validating gRNA efficacy across not just each individual hiPSC line to be evaluated, but also each dCas9 effector to be employed.
