Extended Data Fig. 3: Development of single-AAV CBE strategies.
From: In vivo base editing extends lifespan of a humanized mouse model of prion disease
a, Schematic of single-AAV compatible CBE-mediated stop codon installation strategy and frequency of the desired stop codon installation in HEK293T cells transfected with size-minimized CBEs and corresponding sgRNAs. Four size-minimized Cas9 domain (enCjCas9, evoCjCas9, SauriCas9, and eNme2-Cas9) were fused to TadCBEd and a UGI domain to generate size-minimized CBEs. b and c, Frequency of the desired Q91X edit (b) and R37X edit (c) in HEK293T cells transfected with the base editors and the sgRNAs with the specified modifications. Positions of the poly-U stretch in the enCjCas9 sgRNA scaffold (c) and SauriCas9 sgRNA scaffold (d) are highlighted. ‘No flip’ refers to sgRNA with canonical scaffold; UnF refers to sgRNA with the Un-An position flipped to An-Un. d, Schematic of SauriCas9-TadCBEd, composed of N-terminal NLS (N-NLS), TadCBEd domain, linker 1, SauiCas9 domain, linker 2, UGI domain, and C-terminal NLS (C-NLS). Frequency of the desired R37X edit in HEK293T cells transfected with the SauriCas9-TadCBEd containing the specified modification and the PRNP R37X F-sgRNA. e, Schematic of single-AAV PHP.eB SauriCas9-TadCBEd with PRNP R37X F-sgRNA and single-AAV PHP.eB enCjCas9-TadCBEd with PRNP Q91X F-sgRNA. AAVs (5.1 kb and 5.0 kb, respectively, including ITRs) encode an EFS promoter, TadCBEd deaminase domain, SauriCas9 or enCjCas9 domain, a UGI domain, and a U6 Pol III cassette expressing either the PRNP R37-targeting sgRNA or PRNP Q91-targeting sgRNA. f, Frequency of the specified stop codon installation in the bulk brain hemisphere of Tg25109 mice, 35 days after treatment with either single-AAV PHP.eB SauriCas9-TadCBEd PRNP R37X F-sgRNA (n=6) or single-AAV PHP.eB enCjCas9-TadCBEd PRNP Q91X F-sgRNA (n=5). AAVs were retro-orbitally administered to 5–8 weeks-old Tg25109 mice at a total dose of 1.5x1013 vg/kg. Dots represent individual biological replicates (n=3 unless noted otherwise) and data are presented as mean values +/- 95% CI.
