Fig. 1: Establishment of a benchmarking assay for Cas9 activity.

a Summary of assays discussed in this manuscript and the Cas9 enzymes studied in each assay. HiFi-Cas9 was added at the off-target characterization stage, as our initial clone contained a mutation. b Schematic of the three 5′-types screened. Location of the PAM sequence is indicated in red. c Schematic of PAM-mapping screens. d Replicate correlation (Pearson’s r), calculated from n = 2 experimental replicates for each variant screened. e Example fraction active calculation for WT-Cas9 at NGGN PAMs. f Precision-recall curves for WT-Cas9 and high-fidelity variants profiled with the PAM-mapping library. Guides of all 5′-types are included in this calculation. Dashed lines designate the recall at 95% precision for WT-Cas9. g Recall values at 95% precision for WT-Cas9 and high-fidelity variants profiled with the PAM-mapping library (NGGN PAMs only), discretized by 5′-type. Source data are provided as a Source Data file.