a, Schematic of the genomic sequence surrounding the HCM R403Q pathogenic variant. The ABE protospacer (green line) and PAM (purple line) are underlined. The pathogenic variant R403Q (numbered according to the human MYH7 amino acid residue) is shown with flanking amino acid residues (N, amino terminus; C, carboxyl terminus). R403Q is located at position A5 (blue), and potential bystander edits are at positions 10 (brown) and 11 (orange), numbered from the 5′ end of the protospacer. Bystander editing at each position (arrows) would encode missense amino acids. b, Schematic of the experimental design depicting AAV9 delivery and subsequent evaluation of disease. Dual-AAV9 ABE8e or single AAV9-Cas9 were injected (solid filled arrow, first dose; unfilled arrow, second dose) into R403Q-129SvEv or R403Q-129SvEv/S4 mice at postnatal weeks 2–3. All R403Q-129SvEv/S4 mice consistently develop LVH by 8–10 weeks (gray box), whereas only male R403Q-129SvEv mice show LVH at 20–25 weeks (blue box). Cardiac morphology and function were assessed by echocardiography at 2–4-week intervals. c, Editing efficiency (%) of the targeted pathogenic variant R403Q was based on high-throughput sequencing gDNA from all LV cells, including cardiomyocytes, fibroblasts, macrophages and endothelial cells, after a single dose of dual-AAV9 ABE8e injection (n = 6 males) or in untreated R403Q mice (n = 5 males), quantified by CRISPResso2. gDNA editing efficiency was calculated as: WT nucleotide percentage minus 50% (to measure the editing beyond the heterozygous baseline) and then divided by 50% (to determine the percentage of observed editing out of the theoretical maximum). d, Editing efficiency of the targeted R403Q allele after a single AAV9 ABE8e injection was assessed by sequencing Myh6 cDNA derived from RNA extracted from the LV (n = 5 males, 3 females), RV (n = 4 males, 2 females), LA (n = 2 males, 2 females) and RA (n = 2 males, 2 females) and in five untreated LVs and RVs from male mice. RNA editing efficiency was calculated as: WT nucleotide percentage minus 50% and then divided by 50%. Data are presented as mean values ± s.d.
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