Fig. 5.

Outline of how to create germline zebrafish CRISPR-Cas9 mutants and CRISPants. To create a germline zebrafish CRISPR-Cas9 mutant (follow path of solid lines), guide RNAs (gRNAs) targeting a gene of interest along with Cas9 mRNA or protein are injected into newly fertilized zebrafish embryos. These G₀ injected embryos are grown to adulthood (2–3 months). G₀ adult founders are crossed to wildtype adults, and the G₁ progeny screened for indels. G₁ progeny with indels are grown to adulthood. G₁ adults containing indels are then incrossed and screened for phenotypes. To perform analyses on injected G₀ CRISPant larvae, optimized gRNAs are injected into newly fertilized zebrafish embryos. Injected CRISPant G₀ larvae are then screened for phenotypes days after the injection. CRISPant phenotypes can be verified by generating a stable, germline mutant