Extended Data Fig. 4: Development of ABE-mediated start codon disruption strategy for PrP reduction.
From: In vivo base editing extends lifespan of a humanized mouse model of prion disease
a, Schematic of ABE-mediated start codon disruption strategy highlighting sgRNA spacer sequences and potential bystander sites. Frequency of M1V and bystander edits in HEK293T cells transfected with ABEs and corresponding sgRNAs. b, Schematics of AAV constructs for dual-AAV PHP.eB SpCas9-ABE8e(V106W) with A5 PRNP M1V F+E-sgRNA and single-AAV PHP.eB SauriCas9-ABE8e with A3 PRNP M1V F-sgRNA. c, Frequency of the desired M1V edit, and d, PrP protein level in the bulk brain hemisphere of Tg25109 mice untreated (n=6), or treated with dual-AAV PHP.eB SpCas9-ABE8e(V106W) A5 PRNP M1V F+E-sgRNA (n=5) or single-AAV PHP.eB SauriCas9-ABE8e A3 PRNP M1V F-sgRNA (n=6). e and f, Dose-dependent effects of single-AAV PHPe.B SauriCas9-ABE8e (5.0x1012 vg/kg, 1.5x1013 vg/kg, and 4.5x1013 vg/kg, n=6 per dose) on M1V editing frequency (e) and PrP levels (f) in the bulk brain hemisphere of treated mice. Data for the ‘1.5x1013 vg/kg’ condition correspond to the ‘SauriCas9-ABE8e’ condition in Extended Data Fig. 4d and e. g, Frequency of the desired M1V edit, and h, PrP protein level in the bulk brain hemisphere of Tg25109 mice treated with single-AAV PHP.eB SauriCas9-ABE8e with A3 M1V F-sgRNA with either EFS promoter (n=6) or pCALM1 promoter (n=4) driving the expression of the SauriCas9-ABE8e. Data for the ‘EFS’ condition correspond to the ‘SauriCas9-ABE8e’ condition in Extended Data Fig. 4d and e. i, Representative allele frequency table showing editing outcome after treatment with single-AAV PHP.eB SauriCas9-ABE8e with A3 PRNP M1V F-sgRNA. Incidences where bystander edits occur without on-target editing are highlighted in red box. j, Frequency of bystander editing that leads to N3S, N3D, and N3G mutation in genomic DNA harvested from brain hemisphere of Tg25109 mice treated with single-AAV PHP.eB SauriCas9-ABE8e with A3 PRNP M1V F-sgRNA, harvested 35 days post-treatment (n=4). Dots represent individual biological replicates (n=3 unless noted otherwise) and data are presented as mean values +/- 95% CI.
