(a) Comparison of growth phenotypes measured in replicate screens after 4, 6, or 8 days of growth from t0. Data from Day 4 were used for all subsequent analyses. n = 35,830 sgRNAs; r2 = squared Pearson correlation coefficient. (b) Comparison of relative % knockdown (quantified via RT-qPCR) and mean relative growth phenotype for 10 intermediate-activity constant region variants paired with two targeting sequences against DPH2. Data represent the mean of technical triplicates. (c) Relative activities of constant regions paired with all 30 targeting sequences, ranked by the average strength of each constant region and displayed as rolling means with a window size of 50. (d) Distribution of all pairwise correlations of constant region relative activities within and between gene targets. n = 30 and 1,350 for intra-gene and inter-gene comparisons, respectively; indicated p-values are derived from a two-tailed Student’s t-test; dashed lines in violin plots indicate the distribution quartiles. (e) Relative activity of each indicated target sequence:constant region pair vs. the mean relative activity of the respective constant region for all targets. Growth phenotypes (γ) with the unmodified constant region are indicated in the figure legends. Lines represent rolling means of individual data points.