Figure 1. Design and performance of Cas9 library.

(a) Schematic of library design indicating design of gene-targeting guides, safe-targeting guides, and non-targeting guides. (b) Growth screens were performed in duplicate in three cell lines, and a ricin screen was performed in duplicate in a single cell line. (c) Precision/recall curve for performance on gold-standard essential genes³⁸. These curves graphically display the trade-off between the fraction of genes correctly identified as essential (precision) and the fraction of all essential genes identified (recall). Untreated conditions were compared to plasmid library composition and replicates were combined and analysed with casTLE²⁰. Previous 4-guide duplicate screen in K562 included as reference²⁰. (d) Schematic of nucleotide sugar and n-glycan synthesis genes in ricin screen results. Ricin treated conditions were compared to untreated conditions in K562, and replicates combined and analysed using casTLE²⁰. Blue boxes indicate the gene knockout protected the cell from ricin while red boxes indicate the gene knockout sensitized the cell to ricin. NS indicates non-significance (q>0.1). White boxes indicate that these genes are known to be on-pathway but were not identified as ricin regulators. (e) Quantile-quantile plot showing altered distribution of P values using safe-targeting or non-targeting control in growth screens. P values are calculated from both replicates using casTLE²⁰.