Fig. 3: PsCas9 edits the human genome and shows low off-target activity.

A Western blot analysis using an antibody detecting the FLAG-tag of PsCas9, SpCas9 and FnCas9 expressed in HEK293T cells. GAPDH was used as a loading control. B Heatmap showing the editing efficiency in HEK293T-cells transfected with PsCas9 and sgRNAs with various spacer length (18-30nt) targeting either EMX1a or CD34. The editing efficiency (percent modified reads per sample) was calculated using amplicon sequencing with CRISPresso2 analysis. Data are presented as mean, n = 3. C Maximum activity of sgRNAs (%) targeting EMX1a containing a single mismatch at the indicated position in HEK293T cells. The data was normalized to the sgRNA with a spacer fully complementary to the EMX1a target site. The editing efficiency was calculated with CRISPResso2 analysis of amplicon sequencing data and shown as percent modified reads in each sample. 5’ of sgRNAs contain a G-G mismatch at position 23 that comes from U6 transcription. Data are presented as mean ± SD, n = 3. D, E On-target editing (D) and off-target editing (E) by PsCas9, SpCas9 and FnCas9 with 20 nt and 22 nt spacers at the depicted loci. For the off-target analysis, the sum of editing at three to four off-target sites was used (See Supplementary Fig. 6A). Control samples were untransfected and the editing efficiency was determined using NGS. Data was analyzed by CRISPResso2 and presented as percent modified reads in each sample. Data are presented as mean ± SD, n ≥ 3. F CHANGE-seq read counts after in vitro genome cleavage with SpCas9, PsCas9 or PsCas9 with optimized CHANGE-seq protocol (PsCas9 end-repair) at the HEK4 site. The signal was scaled to the library depth (shown as read counts per million mapped reads). Data are presented as mean ± SD, n = 3. G Bar plot showing the number hits identified with CHANGE-seq using HEK4 sgRNA for SpCas9 and PsCas9. Displayed hits are present in at least one technical replicate (out of three) for each nuclease. Data are presented as mean ± SD, n = 3 (unpaired t test, two-tailed; **P value = 0.0020). H, I Translocation rates for SpCas9 (gray) and PsCas9 (red) between the depicted targeted loci in HEK293T cells. Balanced translocations were measured by custom ddPCR assays detecting the predicted translocation events. Data are presented as mean ± SD, n = 3 (unpaired t test, two-tailed; **P value = 0.0011; ***P value = 0.0001).