Identification of Cas9 off-target sites by GUIDE-seq and quantification of potential Cas9 off-target cleavage sites using rhAmpSeq technology. (A) Sequences of off-target sites identified by GUIDE-seq for 2 gRNAs targeting the NR3C1 locus (gRNA#1, top panel; gRNA#2, bottom panel). The guide sequence is listed on top with off-target sites shown below. The on-target site is identified with a black square. Mismatches to the guide are shown and highlighted in color with insertions shown in gray below. The number of GUIDE-seq sequencing reads are shown to the right of each site. Pie charts indicate the fractional percentage of the total unique, CRISPR-Cas9–specific read counts that are on-target (orange) and off-target (blue). (B) On- and off-target effects were determined by targeted amplification followed by NGS for exon 2–guide 1 and for exon 2–guide 2. Individual gRNAs were delivered into HEK293 cells stably expressing WT-Cas9 (left panels), or delivered into HEK293 cells by complexing to WT-Cas9 protein (middle panels) or HiFi-Cas9 protein (right panels). Pie charts indicate the percentage of on-target effect (in red) and off-target effect (in blue). (C) Editing efficiency was determined using targeted amplification followed by NGS. Exon 2–guide 1 and exon 2–guide 2 gRNAs were complexed simultaneously with either WT-Cas9 (blue bars) or HiFi-Cas9 (red bars) into HEK293 cells. Editing efficiencies were determined for the known on- and off-target sites for exon 2–guide 1 (left top panel), and exon 2–guide 2 (right top panel). In a similar experiment, exon 2–guide 1 and exon 2–guide 2 gRNAs were complexed simultaneously with either WT-Cas9 (blue bars) or HiFi-Cas9 (red bars) into primary human T cells. Editing efficiencies were determined for the known on- and off-target sites for exon 2–guide 1 (left bottom panel), and exon 2–guide 2 (right bottom panel). Data are shown as mean and SD from n = 3 human donors.