Fig. 2: Optimizing correction of the PAH P281L variant in cellulo and in vivo.
From: Improved specificity and efficiency of in vivo adenine base editing therapies with hybrid guide RNAs
a, Hybrid capture sequencing of ONE-seq-nominated sites in treated versus control PAH P281L HuH-7 cells with PAH1 gRNA in combination with ABE8.8 mRNA (n = 3 treated and n = 3 untreated biological replicates), with seven human sites verified to have off-target editing. b, Spacer sequences of PAH1 standard and hybrid gRNAs. rN, ribonucleotide; dN, deoxyribonucleotide; m, 2’-O-methylation; *, phosphorothioate linkage. DNA substitutions are in bold red and underlined. c, A-to-G editing of the PAH on-target site (correction of P281L variant only, or correction plus unwanted nonsynonymous bystander editing) or verified PAH1 human off-target site (PAH1_OT3) in PAH P281L HuH-7 cells treated with PAH1 standard or hybrid gRNA in combination with ABE8.8 mRNA (n = 2–3 biological replicates per condition; exact numbers are provided in the source data). d, Number of sites with a ONE-seq score of >0.01 for each indicated PAH1 standard or hybrid gRNA with ABE8.8 protein. e, A-to-G editing of the PAH on-target site or verified PAH1 human off-target site in PAH P281L HuH-7 cells treated with PAH1 standard or hybrid gRNA in combination with ABE8.8 mRNA (n = 11–12 biological replicates per condition; exact numbers are provided in the source data). f, Number of sites with a ONE-seq score of >0.01 for each indicated PAH1 standard or hybrid gRNA with ABE8.8 protein. g, Total A-to-G editing of the PAH on-target site or any of seven verified PAH1 human off-target sites in untreated versus treated PAH P281L HuH-7 cells with PAH1 standard or PAH1_hyb22 gRNA in combination with ABE8.8 mRNA (n = 11–12 biological replicates per condition; exact numbers are provided in the source data). h, Number of variant (non-reference-genome) sites with a ONE-seq score of >0.01 for each indicated PAH1 standard or hybrid gRNA with ABE8.8 protein. i, A-to-G editing of the PAH on-target site or verified PAH1 mouse off-target site (PAH1_mOT3) in homozygous PAH P281L mice treated with 2.5 mg kg−1 dose of LNPs with PAH1 standard or hybrid gRNA in combination with ABE8.8 mRNA (n = 4–5 animals per group; exact numbers are provided in the source data). In all relevant graphs, means are shown. P values were calculated with a two-sided Mann–Whitney U test.
