Fig. 1: Primer-extension-mediated sequencing (PEM-seq) detects off-target hotspots of CRISPR/Cas9.

a Overview of PEM-seq. To prepare PEM-seq libraries, primer extension generated a copy of template with biotinylated primer followed by bridge adapter ligation and DNA amplification. Gray bars represented bait region while orange ones for captured prey region; “N” indicated random molecular barcode (RMB) in the bridge adapter. Arrows indicated positions and orientations of primers. See supplemental protocols for details. b Circos plots of Cas9:RAG1A libraries. Three biological replicates were showed from outside to inside, and the displayed translocation junctions were 19,494, 16,005, and 18,078. Genome-wide translocation junctions binned into 5-Mb regions (blue lines) were plotted on a log scale. Chromosomes were showed with centromere to telomere in clockwise direction. Red arrow indicated the Cas9:RAG1A cleavage site. Colored lines connected the on-target site to the off-target hotspots. c Zoomed-in view of Cas9:RAG1A translocation junctions (binned into 2 Mb) on chromosome 7. Red arrows indicated identified off-target hotspots. Cen represented centromere and p/q indicated the chromosome arms. The Pearson correlation coefficient was 0.99 between replicates 1 and 2, 0.99 between replicates 1 and 3, 0.98 between replicates 2 and 3. d Scatter plot of Cas9:RAG1A off-target hotspots in 293T, HCT116, K562, and U2OS cells. y Axis showed frequency of each hotspot per 100,000 editing events (indels plus translocation). Red asterisks indicated the off-target hotspots detected by PEM-seq but not by linear amplification-mediated high-throughput genome-wide translocation sequencing. e Venn Diagram showing the overlap for RAG1A off-target hotspots among indicated cell lines. Legend is the same as showed in d