Fig. 6: The optimal, target-matched SpCas9 nuclease, which shows efficient on-target editing and no off-target effects, is identified for each target using a two-step approach.

a Schematic representation of the two-step screening method used on a hypothetical target example. The first panel shows the on- and off-target activity of a set of IFNs with increasing fidelity on a hypothetical target example. The second panel shows the screening method, which identifies the optimal variant for the target without having to test all of the variants. In the first step, a rough on-target screen is performed, where the WT and three selected IFNs, that divide the target ranking range into four approximately proportional sections, are tested. The second step is a fine-tuning on-target screen, that involves the not yet used variants with higher fidelity than the highest ranking active (green) variant from the first screen, and it identifies the target-matched variants (active variants with the highest fidelity). If necessary, two sufficiently active (here their normalized activity is above 50%, but this may depend on the application under consideration) target-matched variants can be screened for the absence of genome-wide off-targets. b The identification of the target-matched variants that provide appropriate editing without any genome-wide off-target is demonstrated on three targets that had been tested in Tsai et al.²⁰. The numbers in the colored Cas protein illustrations indicate the percentage value of the on-target genome modifications normalized to WT (measured by NGS). Colored circles indicate whether a target was edited with (red) or without (green) off-targets in the GUIDE-seq experiment. The total number of off-target sites detected by GUIDE-seq are shown for each target in bar charts on the right side of the panel. Data related to Fig. 8, Supplementary Figs. 10, 11. Target sequences, NGS and GUIDE-seq data are reported in Supplementary Data files 1, 5, 6.