Figure 7
- ID
- ZDB-FIG-220426-59
- Publication
- Cornean et al., 2022 - Precise in vivo functional analysis of DNA variants with base editing using ACEofBASEs target prediction
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- Figure 7
- Figure 7—figure supplement 1.
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(a) Candidate human CVD gene SNV validation workflow. (b) To target the SNVs evoBE4max mRNA was co-injected into the 1 cell stage of the medaka wild-type or myl7::EGFP, myl7::H2A-mCherry reporter strain together with the corresponding target or oca2-Q333 (control) sgRNAs. Individual, imaged, embryos were then further analyzed to determine the rate of C-to-T transversions. (c) Cytosine editing efficiencies are substantial for all candidate genes tested. Data shown in Figure 7—figure supplement 2 was replotted, including all data points from a-d across all target cytosine along the protospacer. Sample numbers: dapk3-P204 (n = 7), ube2b-R8 (n = 5), usp44-E68 (n = 11), and ptpn11-G504 (n = 11). (d) Representative phenotypes of 4 dpf base edited embryos are shown for all four tested candidate CVD genes including oca2-Q333 controls. Top, ventral view, with V = ventricle, A = atrium. (e–g) Confocal microscopy of selected candidate validations in the reporter background. Hearts were imaged in 7 dpf hatched double fluorescent embryos. Images show maximum projections of the entire detectable cardiac volume with a step size of 1 µm. Cartoons (left) highlight the looping defects observed in usp44 and ptpn11 base edited embryos with ventricle-atrium inversion (f) or tubular heart (g). (e’-g’) Imaged embryos were subsequently genotyped and quantified C-to-T transversions for the target codon are shown. Note: due to the inverted nature of the confocal microscope used, raw images display a mirroring of observed structures, which we corrected here for simpler appreciation. Phenotypic analysis of F1 dapk3-P204L (h) and usp44-E68K (i) embryos revealed that homozygous changes at P204L or E68K lead to cardiac malformations with varying degree: looping (h’) and mild looping defects (h’’, i’’’); altered heart morphology (i’’). Bystander edits (hetero- or homozygous, usp44-E68K) lead to additional developmental defects, including brain and eye abnormalities (i’). Scale bar = 100 µm (d, e–i). dpf = days post fertilization.
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