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Figure 2
An overview of multiple-gene knockout technology and the generation of individual homozygous mutants. Cas9 mRNA and five gRNAs were synthesized and co-injected into 1–2 cell stage embryos of wild-type zebrafish. Mosaic F0 were in-crossed to generate mosaic F1 zebrafish. Then, F1 fish were out-crossed with wild-type to generate F2 fish. The gene mutation results of mosaic F1 were identified by sequencing. Heterozygous F3 zebrafish were crossed to generate homozygous mutants.
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