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In vivo CRISPR/Cas9-mediated REEP5 depletion in zebrafish embryos leads to cardiac abnormalities.a Immunofluorescence analysis of REEP5 expression in wild-type zebrafish hearts at 48 and 96 hpf. Co-staining for REEP5 (green), zn-8 (general plasma membrane; red) and DAPI (nuclear; blue) revealed REEP5 expression in both ventricular and atrial cardiac myocytes, with no nuclear or membrane localization evident. Scale, 50 μm. b–e CRISPR/Cas9-mediated depletion of REEP5 with varying gRNA concentrations reveals the contribution of REEP5 to embryonic heart development in zebrafish embryos. Images shown are representative of 331 control, 410 2× REEP5 gRNA, and 296 3× REEP5 gRNA total injected embryos, n = 3 independent biological replicates. Scale, 500 μm. f, g Optical imaging analysis of control and 2× REEP5 gRNA injected embryos shows cardiac looping defects associated with REEP5-targeted gRNA injection. Scale, 10 μm. h Movie analysis of REEP5 gRNA injected embryos shows dyssynchronous atrio-ventricular beating rhythms compared to control zebrafish embryos. Area profiles were smoothened with a Gaussian curve with σ = 0.2 s. i–k Bar graphs showing i phenotypic penetrance represented as percentage, j heart rate represented as beats per minute (bmp), and k atrioventricular beating dyssynchrony represented as percentage from 331 control, 410 2× REEP5 gRNA, and 296 3× REEP5 gRNA total injected embryos, n = 3 independent biological replicates. Asterisks indicate a statistically significant p value in a Tukey’s multiple comparison analysis where *p < 0.05, **p < 0.01, and ***p < 0.001; data are presented as mean ± SEM.
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