Fig. 3: DNA-PKcs inhibition increases the sensitivity of CRISPR off-target detection.

a,b, Plots of MRE11 ChIP–seq enrichment (number of reads within a 1.5-kb window centered at the cut site, per 1 million total reads, that is, RPM) for samples with (y axis) or without (x axis) DNA-PKcs inhibition at all FANCF site 2 (a) or VEGFA site 2 (b) Cas9 target sites in K562 detected from the DNA-PKcs inhibited samples. Each point in the plot (15 in panel a, 178 in panel b) corresponds to a putative target site. Significant differences (P = 0.00081 or P = 4.57 × 10⁻²⁶) between y-axis and x-axis values were determined using two-sided Wilcoxon signed-rank test. c,d, Genome browser visualization of MRE11 enrichment at an on-target (c) and representative off-target (d) position from K562 with Cas9 targeting FANCF site 2, with (red) or without (blue) DNA-PKcs inhibition. e,f, Plots of MRE11 ChIP–seq enrichment for samples with (y axis) or without (x axis) DNA-PKcs inhibition at positions 10 kb downstream from the actual (e) FANCF site 2 or (f) VEGFA site 2 cut sites, to measure background enrichment adjacent to cut sites. MRE11 enrichment with (y axis) versus without (x axis) DNA-PKcs inhibition at the adjacent background locations was not significantly different (P = 0.21 or 0.18), determined using two-sided Wilcoxon signed-rank test. g, Number of discovered off-target sites with (red) or without (blue) DNA-PKcs inhibition for VEGFA site 2. Quantification of Extended Data Fig. 2a. h,i, Number of discovered off-target sites with (red) or without (blue) DNA-PKcs inhibition for VEGFA site 3, FANCF site 2 (h) and HEK site 4 (i) gRNAs. Quantification of Extended Data Fig. 2b. j, Venn diagram illustrating overlap in the identity of Cas9 target sites discovered from samples with DNA-PKcs inhibition (‘DNA-PKi only’; light blue), without DNA-PKcs inhibition (‘no drug only’; light yellow) or found in both samples (‘both’; light green). Four gRNAs were evaluated.

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