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Fig. 2 | Genome Biology

Fig. 2

From: Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases

Fig. 2

Comparisons of the off-target editing activities of WT SpCas9 and three high-fidelity SpCas9 variants at five off-target sites with sgRNAs produced from U3:tRNA–sgRNA constructs and U3:sgRNA constructs. a, c, e Activities of WT SpCas9, eSpCas9(1.0), eSpCas9(1.1), and SpCas9-HF1 for the two off-targets of site 2 (OT2-1 and OT2-2) and the three off-targets of site 6 (OT6-1, OT6-2, and OT6-3) using sgRNAs produced from U3:tRNA–sgRNA constructs (a and c) and for the three off-targets of site 6 with sgRNAs produced from U3:sgRNA-AN19 constructs (e). The off-targets had one (OT2-1 and OT6-1), two (OT2-2 and OT6-2), and three (OT6-3) mismatches (highlighted in red) to sites 2 and 6, respectively. The PAM is shown in blue. The percentage of indels was used to measure off-target editing activity. Two independent replicates were performed. Solid filled columns indicate replicate 1 and pattern filled columns indicate replicate 2. b, d, f Specificities of WT SpCas9, eSpCas9(1.0), eSpCas9(1.1), and SpCas9-HF1 represented as on-target:off-target indel frequency ratios. On-target:off-target ratios were calculated by dividing the on-target indel frequency by the off-target frequency. When off-target activity was undetectable (the threshold of detection was 0.01% of sequencing reads), we set the off-target efficiency to the threshold of detection (0.01%) and these cases are denoted by a triangle

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