Fig. 3: Optimization of CBE strategy for improved potency.
From: In vivo base editing extends lifespan of a humanized mouse model of prion disease
a, Frequency of R37X installation and bystander editing in HEK293T cells transfected with the specified CBEs and PRNP R37X sgRNA. b, Frequency of R37X installation in HEK293T cells transfected with TadCBEd and PRNP R37X sgRNAs with the specified scaffold modifications: canonical (sgRNA), U-to-A flip (F-sgRNA) or U-to-A flip+5-bp stem extension (F+E-sgRNA). c, Schematic of dual-AAV PHP.eB BE3.9max or TadCBEd with PRNP R37X sgRNA. The 5–8-week-old Tg25109 mice were treated retro-orbitally with AAVs at 1.5 × 1013 vg kg−1. d,e, Frequency of R37X installation (d), and PrP level in bulk brain hemisphere (e) of Tg25109 mice untreated (n = 12) or treated with dual-AAV PHP.eB packaging BE3.9max PRNP R37X sgRNA (n = 8), TadCBEd PRNP R37X sgRNA (n = 8) or TadCBEd PRNP R37X F+E-sgRNA (n = 12) at a total dose of 1.5 × 1013 vg kg−1 and harvested 35 d post injection. In d, untreated versus BE3.9max, P = 0.78; BE3.9max versus TadCBEd, P = 0.0012; TadCBEd versus TadCBEd with F+E-sgRNA, P = 0.030; BE3.9max versus TadCBEd with F+E-sgRNA, P < 0.0001. In e, untreated versus BE3.9max, P > 0.99; BE3.9max versus TadCBEd, P = 0.0031; TadCBEd versus TadCBEd with F+E-sgRNA, P = 0.045; BE3.9max versus TadCBEd with F+E-sgRNA, P < 0.0001. f, Frequency of R37X installation in Tg25109 mice 35 d (n = 8) versus 100 d (n = 6) post treatment with BE3.9max (P = 0.0042) or TadCBEd (P = 0.038). g, Scatter plot showing the relationship between R37X editing frequencies and PrP protein levels in brain tissues of Tg25109 mice treated with BE3.9max or TadCBEd, harvested at 35 d (n = 8) or 100 d (n = 6). Linear regression is shown with solid line and shaded area represents 95% CI. Dots represent individual biological replicates (n = 3 unless noted otherwise) and data are presented as mean ± 95% CI. Significance in d and e was calculated by two-way analysis of variance (ANOVA) test with Bonferroni correction; NS, P > 0.12; *P ≤ 0.033; **P ≤ 0.002; ****P ≤ 0.0001. Significance in f was calculated by two-tailed Student’s t-test; NS, P > 0.05; *P ≤ 0.05; **P ≤ 0.01. kb, kilobases.
