Extended Data Fig. 7: Assessment of in vivo off-target indel mutations induced by gMH–Cas9.

Indel mutation frequencies determined by targeted amplicon sequencing (using high-throughput sequencing) are presented as heat maps for the gMH–Cas9 on-target site (black square) and 63 off-target sites identified from CIRCLE-seq experiments. Each locus was assayed in n = 3 biologically independent mice (labelled 1, 2 and 3) using genomic DNA isolated from the liver of wild-type and knock-in mice treated with experimental adenoviral vector that encodes gMH–Cas9 (gRNA +) or control adenoviral vector GFP–Cas9 (gRNA −). For each site, mismatches relative to the on-target site are shown with coloured boxes and bases in the spacer sequence and are numbered from 1 (most proximal to the PAM) to 20 (most distal from the PAM). The number of read counts found for each site from the CIRCLE-seq experiments on wild-type and knock-in mouse genomic DNA are shown in the left columns (ranked from highest to lowest based on counts in the wild-type genomic DNA CIRCLE-seq experiment). Each box in the heat map represents a single sequencing experiment. Sites that were significantly different between the experimental (gRNA +) and control (gRNA −) samples are highlighted with an orange outline around the boxes. Additional closely matched sites in the mouse genome (not identified from the CIRCLE-seq experiments) that were examined for indel mutations are boxed in red at the bottom of the figure. See Supplementary Table 7 for source data and P values (negative binomial).