Library 2 Sublibrary A: Analysis of 2,391 6-plex constructs with sufficient read coverage and that encode in the test position one of 99 spacers that scored as strong hits as single crRNAs in the Library 1 screen (orange boxplots), or negative control crRNA (grey boxplots). Boxplots show cell fitness scores averaged from the top three context constructs of each test position spacer in the 6-plex array from two screen replicates. Recall is calculated as the percentage of essential TSS-targeting spacers (that were empirically active in the single crRNA Library 1 screen) with a cell fitness defect in the Library 2 6-plex crRNA array screen for a given test position, using the 5th percentile of constructs containing negative control spacer in the same test position as a threshold (dashed line) for calling hits. Boxplots display median, interquartile range, whiskers indicating 1.5x interquartile range, and outliers. One-sided Wilcoxon rank-sum test was performed for the difference in the distributions of negative control spacers ( n = 69–506 constructs) versus TSS-targeting spacers ( n = 20–99 constructs) at each position, with asterisks indicating P < 0.01.
