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Figure 3 Zebrafish as discovery platform for congenital heart disease genes. General workflow for linking patient mutations to cardiac phenotypes using zebrafish. (A) Whole-genome sequencing or other approaches inform about possible candidate genes responsible for congenital heart anomalies. (B) CRISPR-Cas9-based mutagenesis of the candidate genes by microinjection enables the generation of germline mutations towards isolating mutant alleles, as well as first phenotype assessment and screening in F0 embryos (crispants). (C) Establishment of germline mutants for subsequent genetic follow-up of phenotypic consequences of candidate mutations. (D) Phenotype assessment of crispants or germline mutants via microscopy, assisted by transgenic labeling or staining for individual gene expression patterns (i.e., mRNA in situ hybridization, immunohistochemistry, and others). The fluorescent panel is a max projection of a triple transgenic zebrafish at 3 dpf carrying drl:EGFP, myl7:CFP, and lmo2:dsRed [24,88,198,199]. See text for details. Schematics generated with BioRender.