Figure 1. An unbiased CRISPRi screen identifies genetic targets of alendronate.

(A) Schematic illustrating the workflow of the genome-wide CRISPRi screen. The IC₅₀ of alendronate in K562 cells is 250 μM. (B) Volcano plot showing, for each gene, a ρ score that averages the normalized fold enrichment (in the treated population compared to the untreated control) of the gene’s three most effective sgRNAs, and a Mann-Whitney P-value for fold enrichment (Gilbert et al., 2014). The dashed lines represent thresholds used to identify significant hits. Positive ρ scores correspond to resistance hits and negative scores to sensitizing hits. (C) Gene names and annotated functions of the top seven resistance and sensitizing hits. Genes are sorted by the absolute values of their ρ scores in descending order. SLC37A3 is marked in bold. (D) Diagram of the mevalonate pathway, with genes in the pathway that were identified as significant hits marked with their ρ scores. Resistance hits are color-coded in red and sensitizing hits in blue.

Figure 1—figure supplement 1. Additional analysis of the whole-genome CRISPRi screen.

(A) Evaluation of the reproducibility of the CRISPRi screen. The enrichment score (ρ) of each sgRNA was calculated separately from two biological replicates of the CRISPRi screen and compared in a scatter plot. Data points corresponding to negative control sgRNAs are colored in gray. (B) Quantile-quantile plot comparing the distribution of observed average sgRNA enrichment scores (ρ scores) of each gene in the genome with a Gaussian distribution that has the same mean and standard deviation. The dashed gray line represents the predicted location of data points if the distribution of ρ scores is indeed Gaussian. The large deviations from the gray line observed at the two ends of the distribution indicate that the silencing of those genes has stronger effects than expected by pure Gaussian noise and is therefore likely to be biologically meaningful. The dotted lines are arbitrary thresholds set to select resistance hits (red dotted line) and sensitizing hits (blue dotted line) that deviate significantly from Gaussian predictions. 398 resistance hits and 28 sensitizing hits passed the thresholds. (C–D) Gene Ontology (GO) pathway enrichment analysis of the top 100 resistance hits (C) and the top 30 sensitizing hits (D) identified in the CRISPRi screen. Only the most specific subclasses that are statistically significant are shown. Both fold enrichment of pathway genes and P-values of fold enrichment are displayed. Fold enrichment values were clipped at 100 fold. P-values were corrected for multiple testing using Bonferroni correction. Note that genes involved in the mevalonate pathway, which includes IPP biosynthesis, geranyl phosphate synthesis and farnesyl phosphate synthesis, are significantly enriched in top hits from the screen. IPP: isopentenyl pyrophosphate. (E) Volcano plot showing the results from a second CRISPRi screen using zoledronate, another representative N-BP, as the selection agent. Plot layout is the same as in Figure 1B. SLC37A3 and ATRAID are highlighted in red and cyan, respectively. Significant hits were defined as genes that had a fold enrichment with an absolute value larger than 0.1, and a P-value smaller than 0.05.