(a) Representative plots illustrating gating strategy to select cells for analysis. (b) Comparison of relative activities obtained from two replicate transductions. Relative activity was defined as the fold-knockdown of each mismatched variant (GFPsgRNA[non-targeting]/GFPsgRNA[variant]) divided by the fold-knockdown of the perfectly-matched sgRNA. The background fluorescence of a GFP– strain was subtracted from all GFP values prior to other calculations. n = 57 sgRNAs; r2 = squared Pearson correlation coefficient. (c) KDE plots of GFP distributions 10 days after transducing K562 GFP+ cells with the perfectly-matched sgRNA, a non-targeting sgRNA, and each of the 57 singly-mismatched variants. Fluorescence of GFP– K562 cells is shown in gray. Although most GFP distributions are unimodal, some are broadened compared to those with the perfectly matched sgRNA or the negative control sgRNA. This heterogeneity could be a consequence of the random integration of the GFP locus, cell-to-cell differences in expression of the dCas9-KRAB effector in our polyclonal cell line, the amplification of gene expression bursts by long GFP half-lives, or a combination of these factors. Two replicate transductions were evaluated for each sgRNA (see panel b); data from one replicate are shown here.