Fig. 4: Off-target analysis of R37X base editing strategy in human and mouse genome.
From: In vivo base editing extends lifespan of a humanized mouse model of prion disease
a, The percentage of C•G-to-T•A substitution in BE-AAV-treated samples above background (untreated samples) at 299 CIRCLE-seq nominated off-target sites in the human genome (GRCh37). Genomic DNA was extracted from HEK293T cells untreated (n = 3) or after 3 d following transfection of plasmids encoding TadCBEd and the PRNP R37X sgRNA (n = 3). Each dot represents mean of three biological replicates. b,c, The percentage of C•G-to-T•A substitution in BE-AAV-treated samples above background (untreated samples) at the top 100 CIRCLE-seq-nominated off-target sites in the mouse genome (GRCm38). Genomic DNA was extracted from the bulk brain hemisphere of Tg25109 mice untreated (n = 6), or 35 d (b) and 100 d (c) after treatment with dual-AAV PHP.eB BE3.9max with PRNP R37X sgRNA (n = 6), or dual-AAV PHP.eB TadCBEd with PRNP R37X sgRNA (n = 6) at a total dose of 1.5 × 1013 vg kg−1. Each dot represents mean of six biological replicates. d, The percentage of C•G-to-T•A substitution in BE-AAV-treated samples above background at the top 100 CIRCLE-seq-nominated off-target sites in the mouse genome (GRCm38). Genomic DNA was extracted from the bulk brain hemispheres of Tg25109 mice untreated (n = 5), or 600 d after treatment with dual-AAV PHP.eB BE3.9max with PRNP R37X sgRNA (n = 5) at a total dose of 1 × 1014 vg kg−1. Each dot represents mean of five biological replicates. In all panels, significance was calculated by one-tailed Student’s t-test. Off-target editing with P > 0.01 compared with untreated control is labeled with hollow circles, and those with P ≤ 0.01 are labeled with solid circles. Plots showing individual data points and error bars are provided in Supplementary Figs. 1–4.
