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Figure 5
(a) Schematic diagram of protocol after co-injection of cytosine editor mRNA (comparing BE4-Gam, ancBE4max, evoBE4max) with sgRNA tnnt2a-Q114 (PTC) into one-cell stage medaka embryos; control injections only contained the sgRNA. (b) Editing of tnnt2a resulted in a range of phenotypes classified into five categories, including general morphogenic (global), dysmorphic but still functional heart chambers (cardiac), and non-contractile hearts (silent), where cardiac and silent phenotype groups displayed homogeneously additional developmental retardation. Scale bar = 400 µm (overview) and 100 µm (zoom-in). Ventricle (V), atrium (A), diastole (Dia) and systole (Sys) are indicated. (b') Representative scheme of fractional shortening of the heart chambers in specified phenotype groups highlighting significant morphological consequences (small ventricle) in the silent heart group. (c) Fraction of phenotype scores as a consequence of cytosine base editor injections. (d) Summary of editor type-specific C-to-T conversion efficiencies relative to the target C protospacer position grouped by phenotype class for BE4-Gam (n = 6), ancBE4max (n = 6) and evoBE4max (n = 12). To highlight the dinucleotide context, the nucleotide preceding the target C is shown by red (A), green (T), blue (C), and yellow (G) squares below the respective C. (d') Example Sanger sequencing reads of single edited embryos with resulting missense and stop-gain mutations through editing at C5, C7, and C8, respectively (e) Amplicon-seq of the target region of a subset (n = 5) of evoBE4max edited gDNA samples (single embryos) quantified target C8-to-T editing as well as indel frequencies. Aligned Illumina-reads analyzed, 7094 (control); 11,557 (evoBE4max rep1); 2561 (evoBE4max rep2); 2481 (evoBE4max rep3); 37,751 (evoBE4max rep4); 48,791 (evoBE4max rep5). (f) Phenotypic analysis of F1 tnnt2a-Q114X mutants revealed complete penetrance (n = 5) of the silent heart phenotype with same phenotypic profile as for F0 edits. dpf = days post fertilization.
Stop-gain mutation in medaka troponin T gene by cytosine base editing accurately recreates mutant phenotypes in F0.