Fig. 1

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.