S2A) and qPCR (Figures S2B and S2C) data suggest that the difference in dCas9 expression across individuals is small, an observation that is consistent with findings that dCas9−KRAB expression does not correlate to the level of changes gene expression achieved (Dixit et al., 2016). The generation of antibiotic selection of stable lines expressing dCas9−VPR, dCas9−KRAB, and dCas9-VP64 sometimes, but not always, improved gRNA efficacy in our hands. Owing to high transduction efficiencies observed with our (small) gRNA expression vectors, we did not test the impact of gRNA selection; nonetheless, it is possible that gRNA selection is a critical variable and/or that the timing of selection is important such that dCas9-effector and gRNA selection is more appropriately conducted concurrently instead of sequentially. More likely, the solution may simply entail multiplexing a variety of gRNAs simultaneously (Figures 4E and 5B).
