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Fig. 4 Multiplex mutagenesis for duplicated genes and synthetic rescue using F0 approach. (A) Schematics of gRNA target loci (blue triangle), gene exons (gray boxes) and their corresponding kinase domain (yellow boxes) for fgfr1a, fgfr1b and fgfr2 are shown. gRNA_1 and gRNA_2 target the beginning of the gene, while gRNA_3 targets the exon encoding the kinase domain. Editing efficiency for each gRNA was shown below. Representative phenotypic images for (B) kinked tail, (C) defective pectoral fins, and (D) abnormal otoliths of F0 animals at 3 dpf, along with their quantification results for different gRNA combinations, are presented. For phenotype categorization, animals showing either a kinked tail (indicated by red arrow in B), complete absence of both fins (C), or two otoliths stuck together (D) are considered in the strong category. (E) Schematics of gRNA target loci, exons, and corresponding functional domains for both llgl2 and erbb2 are depicted. The exons encoding LLGL domain for llgl2 and the kinase domain for erbb2 are shown in yellow boxes. (F) Editing efficiency for each gRNA. (G–J) Representative images for uninjected, llgl2 F0, erbb2 F0 and llgl2;erbb2 F0 at 5 dpf. The white arrow indicates the beginning position of the dorsal fin fold observed in erbb2 F0 knockouts, and red arrowheads indicated the uncontrolled proliferation of epidermal cells. The lower jaw image was enlarged in (G’-J’). Because these phenotypes are difficult to quantify the severity, animals showing excessive epidermal cells were counted with phenotype for llgl2 F0 knockouts, and animals showing aberrant dorsal fin fold and missing lower jaw were counted as phenotype for erbb2 F0 knockouts. For the llgl2;erbb2 F0 group, animals showing either excessive epidermal cells or aberrant dorsal fin fold or missing lower jaw were counted as phenotype. We didn’t see any dysmorphic (phenotypes other than characterized) animals in these experiments. (K) Quantifications of animals with phenotypes as shown in (G-J).