Fig. 2

Low-specificity sgRNAs confound identification of essential motifs in dense-tiling screen of loop anchors and enhancers. a A dense-tiling Cas9 growth screen was performed with sgRNAs densely tiling two types of regions: (1) 1 kb windows around select hit and non-hit CTCF loop anchors from the CTCF motif screen and (2) two enhancers of GATA1, previously called eGATA1 and eHDAC6. b As a positive control, we verified that the dense-tiling screen correctly maps the boundaries of exons of essential genes with high-specificity sgRNAs. Each point is the average enrichment of two biological replicates and the bar is the standard error. c Dense-tiling screen results from a 1 kb region centered on a motif that was a false positive hit in the original motif-targeting screen (targeted with sgRNAs 15776 and 15777 and also shown in Fig. 1 and Supplementary Fig. 1). All evidence for the essentiality of a CTCF motif comes from low-specificity sgRNAs. Motifs in ChIP-seq peaks are shown as black boxes and CTCF motifs as green boxes. d Dense-tiling screen results from two regions containing enhancers of the essential gene GATA1. sgRNAs selected for validation studies are labeled (e.g., 1 L represents the first sgRNA with a low specificity score). ChromHMM is colored according to the 15-state scheme⁷⁶ (briefly, reds are predicted promoter states, yellows are enhancer states, and greens are other transcriptionally active states). e The enhancer motif-targeting sgRNAs identified in d do not significantly decrease GATA1 expression according to qPCR (p > 0.05, ANOVA). Each dot is a sgRNA infection biological replicate. f The sgRNAs identified in d do not significantly decrease GATA1 protein expression according to Western blot. g The sgRNAs identified in d do not significantly decrease GATA1 protein expression according to flow cytometry for GATA1 protein level. Additional validation data are shown in Supplementary Fig. 4. Source data are available in the Source Data file