Fig. 2: Generation of Hifi-DdCBE for inducing m.G11185A base editing on MT-ND4 gene and successful manufacturing of MT-ND4 mutant mice.
From: In vivo mitochondrial base editing restores genotype and visual function in a mouse model of LHON
a Schematic illustration of Hifi-DdCBE in mouse MT-ND4 gene. Base editing window is annotated with black, and the TALE binding sequence with gray. Desired editing locus was shown in red (m.G11185A). b Editing efficiency of m.G11185A in buffer injected blastocysts (n = 5), Hifi-DdCBE injected blastocysts (n = 15), and Hifi-DdCBE injected pups (n = 8). The exact p-values were 7.5902E-13 in buffer injected blastocysts and Hifi-DdCBE injected blastocysts, and 9.87332E-11 in Hifi-DdCBE injected pups. (***p < 0.001 using Student’s two-tailed t-test). Bars indicate median efficiency in each groups. c MT-ND4 gene mutation alleles induced by Hifi-DdCBE in blastocysts. Yellow bars indicate the frequencies of m.G11185A without undesired editing. Desired editing locus was shown in red (m.G11185A), and undesired editing loci (m.G11182A and m.C11188T) were in blue. d MT-ND4 gene mutation alleles induced by Hifi-DdCBE in pups. Yellow bars indicate the frequencies of m.G11185A without undesired editing. Desired editing locus was shown in red (m.G11185A), and undesired editing loci (m.G11182A and m.C11188T) were in blue. e Proportion of MT-ND4 gene mutation alleles induced in DddA11 injected blastocysts (n = 8), Hifi-DdCBE injected blastocysts (n = 8), and Hifi-DdCBE injected pups (n = 5). Alleles are distributed into 4 parts: WT (white), m.G11185A only (sky blue), m.G11185A with m.G11182A and m.C11188T (navy), and only m.G11182A and m.C11188T editing frequencies (gray). f Mitochondrial genome-wide off-target editing in buffer injected blastocyst, Hifi-DdCBE injected blastocysts, and Hifi-DdCBE injected pups. Only G11185A frequencies shown in blue, G11185A with m.G11182A and m.C11188T in red, and off-targets were in black. g Relative ratio of mitochondrial genome-wide off-target activities occurring outside of editing range in buffer injected blastocysts (n = 2), DddA11 injected blastocysts (n = 6), Hifi-DdCBE injected blastocysts (n = 5), and Hifi-DdCBE injected pups (n = 3). The exact p-values were 0.0017 in DddA11 injected blastocyst and Hifi-DdCBE injected blastocyst, 0.0007 in DddA11 injected blastocyst and Hifi-DdCBE injected pup. Error bars indicate standard error of the mean (s.e.m.) for biologically independent samples. (**p < 0.01, ***p < 0.001 using Student’s two-tailed t-test).
