Fig. 1: Effect of DNA-PKcs inhibition on DNA repair at CRISPR–Cas-targeted locations.
From: Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+
a,b, Schematic of genome-wide CRISPR off-target detection using MRE11 ChIP–seq. a, Cells are unsynchronized, so only some cells have MRE11 at Cas9 cut sites at a given time (DISCOVER-Seq). b, Inhibition of NHEJ directs DNA repair to slower, MRE11-dependent pathways. c, Effect of repair factor inhibition on MRE11 residence at the VEGFA site 3 on-target site, measured by ChIP–qPCR estimating ‘reads per million’ (RPM) enrichment at 12 h after Cas9 delivery in HEK293T cells. Each point corresponds to a different biologically independent replicate of a sample exposed to the DNA repair inhibitor listed in the x axis. Red line is the mean of two biologically independent replicates. Samples with DNA-PKcs inhibition (n = 4) have significantly higher estimated RPM compared with samples without DNA-PKcs inhibition (n = 8) using two-sided Student’s t-test (P = 9.65 × 10−5). d, Increased MRE11 residence upon DNA-PKcs inhibition using Ku-60648 (red) versus without inhibition (blue), measured by ChIP–qPCR. Measured over multiple time points (4 h, 12 h, 24 h) after delivery of Cas9 targeting VEGFA site 2 in HEK293T (left plot), with Cas9 targeting FANCF site 2 in K562 at 12 h (middle plot) and with Cas9 targeting HEK site 4 in HEK293T at 12 h (right plot). Plots display the mean over two biologically independent replicates for left and middle plots, and one biologically independent replicate for the right plot. e, Plot of estimated RPM enrichment normalized to the no drug sample from data in panel d, for sample pairs with (‘Ku-60648’) or without (‘no drug’) DNA-PKcs inhibition. Normalized RPM enrichment with DNA-PKcs inhibition was significantly higher than without inhibitor (P = 0.0001), using two-sided Wilcoxon signed-rank test. Red line indicates mean of n = 14 total samples pooled from panel d; green points are HEK293T, VEGFA site 2; purple points are HEK293T, HEK site 4; red points are K562, FANCF site 2. ***P < 0.001.
