Fig. 2. Cleavage activity at off-targets modelled with binding free energy changes.

a Sequences and properties of off-targets identified by GUIDE-seq³⁰ for four gRNAs that have at least one off-target cleaved more efficiently than the on-target. The sequence of the on-target site is reported on top of a list of the profiles of targets detected by GUIDE-seq. Each sequence in the listed targets is shown as follows: dots indicate bases that are the same as the on-target site, while differences between the on-target sequence are displayed with coloured nucleotides. Off-targets were filtered to avoid sites with >3 mismatches to the gRNA, or mismatches located in the four PAM-proximal nucleotides or the PAM itself. b Histogram of GUIDE-seq read counts at target sites in the full dataset of Tsai et al. A red vertical bar indicates the arbitrary threshold of 300, used to distinguish sites with low off-target activity. c gRNA–DNA hybridisation free energy change ΔG_H of target sites grouped by cleavage activity. The sweet spot preferential range of hybridisation free energy change ΔG_H, which contains 80% of the highly efficient gRNAs (top 20% efficient) in the merged dataset of Xiang et al., used for free energy change profiling, is highlighted in grey. d Percentage of G and C bases at target positions complementary to the gRNA. Target sites are grouped by cleavage activity. The preferential range of GC%, defined as in (c), is highlighted in grey. Source data are provided as a Source Data file.