Fig. 2

Quantitative analysis of DNA repair outcome of Cas9-induced DNA double-strand break in hESCs. a Schematic representation of the experimental design. Cas9 RNPs designed to cleave the first exon of PANX1 were electroporated to H1 hESCs. IDMseq was used to analyze the locus in edited hESCs 48 h later. b Large SVs detected by IDMseq and VAULT in edited hESCs. Five SV groups were shown with deletion length ranging from 419 to 5494 bp. The red dotted line represents the Cas9 cutting site. The coverage of Nanopore reads is shown on top of each track in gray. The colored lines on the left side of the coverage plot represent the UMI for the group. Individual Nanopore reads within the group are shown under the coverage plot. c The frequency of deletions or insertions of different size detected in Pan1-edited hESCs. Certain deletions and insertions occur at disproportionally high frequencies. For example, a 5494-bp deletion was found in 56 UMI groups, which indicates a possible hotspot of Cas9-induced large deletion. d Distribution of SNVs detected by IDMseq and VAULT in Pan1-edited hESCs. Somatic SNVs are shown in green, while the cell-line specific SNVs are shown in red (using 40 bp of bin size in the figure). Somatic SNVs cannot be detected if variant calling is done en masse without UMI analysis (see the coverage track). Cell-line specific SNVs are detected in ensemble analysis (see colored lines in the coverage track) and most of them have been reported as common SNPs in dbSNP-141 database (common SNP track). The Cas9 cut site is indicated by the red triangle. e The number of presumed somatic SNVs per Mb (y-axis) in PANX1 WT and Cas9-edited cells. f Analysis of somatic mutations detected in Pan1-edited hESCs based on functional annotation and base change. The majority of base changes are G to A and C to T