Fig. 1: Development of initial base editing strategies to install stop codon in PRNP locus.
From: In vivo base editing extends lifespan of a humanized mouse model of prion disease
a, Schematic of the CBE-mediated stop codon installation as a strategy to knockdown cellular PrP. The PRNP locus consists of N-terminal (dark blue, amino acids 1–144) and C-terminal (light blue, amino acids 145–253) domains. The signal peptide (gray, amino acids 1–22), octapeptide repeat (OPR) region (dashed box, amino acids 51–90) and GPI signal (gray, amino acids 231–253) are highlighted. CBE may convert CAG (Gln), CAA (Gln), CGA (Arg) or TGG (Trp) codons to a stop codon. sgRNA spacers that install stop codons in PRNP evaluated in this study are shown as half-arrows. Truncated PrP no longer templates fibril formation. b, Frequency of the desired stop codon installation or indel formation from candidate sgRNAs using BE4max via plasmid transfection of HEK293T cells. c, Editing efficiency at bystander positions with BE4max and PRNP R37X sgRNA via plasmid transfection of HEK293T cells. Three silent mutations (G35G, S36S and Y38Y) are possible due to bystander editing from cytosine base editing. d, Schematic of dual-AAV PHP.eB BE3.9max with PRNP R37X sgRNA. The N-terminal AAV encodes a Cbh promoter, APOBEC deaminase domain and amino acids 1–572 of SpCas9 fused to NpuN intein. The C-terminal AAV encodes a Cbh promoter, NpuC intein, amino acids 573–1367 of SpCas9 and one copy of the UGI domain. Both AAVs contain a U6 Pol III cassette expressing the PRNP R37-targeting sgRNA. e, Experimental design for initial assessment of the effect of PRNP base editing on PrP levels. The 5–8-week-old Tg25109 mice were treated retro-orbitally with dual-AAV PHP.eB BE3.9max for installation of PRNP R37X at a dose of 1 × 1014 vg kg−1. Brain was harvested 100 d post injection to assess editing efficiency via HTS and PrP protein reduction via ELISA. f, Frequency of R37X installation in untreated (n = 3) and dual-AAV PHP.eB BE3.9max-treated mice (n = 3). g, PrP levels in dual-AAV PHP.eB BE3.9max-treated mice (n = 3) in the bulk brain hemisphere normalized to those of untreated mice (n = 3). Dots represent individual biological replicates (n = 3) and data are presented as mean ± 95% CI.
