Overview of mutagenesis and phenotyping strategies. (A) Single-guide RNA (sgRNA) was synthesized from a template that is generated by annealing and extending oligo A and B. Oligo B is generic and is common in all reactions, while Oligo A contains a T7 promoter, 20-nt target sequence, and another 20 nt overlapping the chimeric gRNA core sequence. Two sgRNAs targeting a single gene were co-injected along with Cas9mRNA into either the yolk or the cell of one-cell stage embryos. The injected embryos were raised to generate the founder fish. (B) The founder fish were then outcrossed to wild type to generate heterozygous F₁ fish. The mutant fish were identified by fluorescence PCR and sequencing. The siblings carrying mutations were then crossed to generate F₂ progeny, and phenotype-genotype correlations were done using the F₂ embryos. (C) Alternatively, the founder fish were inbred and phenotyping was performed in the F₁ generation, and the embryos were genotyped by fluorescence PCR or sequencing. (D) Phenotypes can also be observed in the injected embryos from 0 to 5 d, although off-target effects are more common with this approach. In order to score phenotypes in injected embryos, the sgRNA and Cas9 must be injected in the cell instead of the yolk to achieve maximum efficiency.

fgfr4^-/- phenotype identified by inbreeding injected founder fish. Two single-guide RNAs (sgRNAs) targeting two different exons of the fgfr4 gene were co-injected with Cas9mRNA into wild-type embryos and raised to adults to generate founder fish (F₀). Six pairs of founder fish (F₀) were inbred and scored for mutant phenotypes. One of the six pairs showed multiple embryos with the same phenotype observable at 48 h post-fertilization. (A) Wild-type embryo at 48 h post-fertilization. White arrow indicates a normally sized hindbrain, white arrowhead indicates a normal heart chamber, and black arrow represents a normal tail length. (B) An fgfr4 compound heterozygous mutation displaying multiple phenotypes. White arrow indicates a reduced hindbrain region, white arrowhead points to a heart edema, and black arrow shows the shortened body axis. All phenotypes were only found in the compound heterozygous mutant embryos.

Biallelic disruption of tyrosinase, chordin and fgf-24 by CRISPR/Cas9. Two targets were co-injected with Cas9 in the cell of 1-cell stage of embryos and the phenotypes were observed within 5 days of development. Lateral views (A-B) and Dorsal view (C) of the wild-type embryos. (D) tyrosinase CRISPR-injected embryos showed reduced pigmentation, (E) chordin CRISPR-injected embryos showed dorsalization of ventral tissues and (F) fgf24 embryos showed lack of pectoral fins.