Fig. 9: Correcting a clinically relevant mutation without off-target cleavage using the two-step screening method.

a The strategy to correct the mutation causing Xeroderma pigmentosum in patient-derived fibroblast cells is shown, including the sequence environment of the mutation (disease-causing mutation – red letter, WT nucleotide– green letter, silent mutation – blue letter). b By using the two-step screening method we identified B-HypaSpCas9 to be used for editing without any detectable genome-wide off-target. Values in the colored Cas protein illustrations indicate the percentage value of the on-target genome modifications normalized to WT (measured by NGS). Hypa-R being a non-Blackjack-IFN exhibited diminished (<0.2) activity with the 21G-sgRNA (data not shown here, data available in Supplementary Data file 5). Colored circles indicate whether a target was edited with (red) or without (green) off-targets in the GUIDE-seq experiment. c Bar chart showing the total number of off-target sites detected by GUIDE-seq. ‘*’ indicates that no off-target was detected in a repeated GUIDE-seq experiment, even though the read numbers were higher in the repeated experiment (see Supplementary Fig. 11). d B-HypaSpCas9 with single strand oligo nucleotide repair using HDR enhancer M3814⁸⁶ provides WT-like level of correction of the R683W (2047C>T) mutation. Means and SD are shown; n = 3. a–d 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.