Fig. 4: In vivo gene editing with PsCas9 in the human PCSK9-KI mouse model.

A Experimental setup for the in vivo workflow. Humanized PCSK9-KI mice were injected with adenovirus expressing PsCas9 and gMH sgRNA. After 1 week, liver tissue was sampled to estimate DNA editing activity. On-target editing was analyzed by targeted amplicon sequencing, off-target editing by Duplex-Seq. B The editing efficiency at mouse Pcsk9 and human PCSK9 elicited by PsCas9 and SpCas9 as determined with amplicon sequencing followed by CRISPResso2 analysis is shown as percentage of indel-containing reads in each sample. Graphs show individual values, as well as mean ± SEM from n = 3 biologically independent mice. A two-way ANOVA followed by Sidak’s multiple comparison test was performed to evaluate statistical significance (**P value = 0.009; ns P value = 0.1846). C Gene editing at off-target sites assayed by Duplex-Seq followed by CRISPResso2 analysis. Heatmaps present editing efficiency for the two target sites (highlighted in green), as well as the five off-target sites detected in SpCas9-treated mice (Fig. 1C). Duplex-Seq was performed with n = 3 biologically independent mice (1, 2, 3) using genomic DNA isolated from the liver of mice treated with adenoviral vector expressing sgRNA gMH together with PsCas9. Adenoviral vector encoding SpCas9 without an sgRNA was used as a control. Values show editing data as percent indel-containing reads normalized to controls. An extended table showing non-normalized data of all assayed off-target sites, as well as the two on-target sites can be found in Supplementary Fig. 3. The detection limit reached in this experimental set-up was 0.01%. All Duplex-Seq experiments were performed in parallel in the same experiment. D Schematic of using Duplex-Seq to assay indels and junction reads that report on translocation frequencies. E, F Evaluation of translocations between the two PCSK9 on-target sites in vivo. Translocation percentage shown as the fraction of translocated sequencing reads divided by all human (E) or all mouse on-target reads (F). Graphs show individual values, as well as mean ± SD from n = 3 biologically independent mice. Unpaired, two-tailed t test was performed to evaluate statistical significance (***P value = 0.0010 (E); ****P value < 0.001 (F)). Note that all four possible translocation outcomes between the two genes are included in the analysis.