Fig. 1

GuideScan specificity filtering of a genome-scale CRISPR-Cas9 screen for essential CTCF loop anchors. a Schematic of CTCF loop anchor motif screen, with 2 to 5 sgRNAs targeting each CTCF motif. b Fitness effects are reproducible between independently transduced biological replicates of the screen. sgRNAs targeting essential gene exons or the BCR-ABL amplification drop out during the growth screen, as expected. Guide enrichment values are the log₂(fold-change) of an sgRNA’s sequencing counts from after the screen compared with the original plasmid pool, computed with the casTLE screen analysis software⁵. c The growth effects of CTCF motif-targeting sgRNA are validated in individual competitive growth assays after lentiviral delivery of single guides to K562-Cas9 cells. Error bars are standard deviation of three technical replicates. d Comparison of sgRNA fitness effects with the number of off-target sites with 2-3 mismatches. Any sgRNAs with off-target sites with only 0 or 1 mismatch, as determined by the GuideScan search algorithm, are excluded. e Low-specificity score guides are significantly enriched among CTCF motif-targeting guides with fitness effects. The Fisher’s exact test provided the p-value for the association between fitness effect and specificity using the 2 × 2 contingency table of the numbers of guides in each quadrant based on the thresholds drawn in black lines. Numbers in corners correspond to the number of CTCF site-targeting guides (blue circles) in the quadrant. The off-target search was done with GuideScan, which retrieves all off-target locations with 2 or 3 mismatches to the sgRNA spacer. sgRNAs with >1 perfect matches to the genome or >0 off-target locations with only 1 mismatch are not searchable within the GuideScan trie data structure and were excluded from this analysis. f Filtering for high-specificity scores removes all CTCF motifs with concordant evidence of fitness effects from multiple sgRNAs. Gray circles are screen biological replicates. Source data are available in the Source Data file