Fig. 3

CRISPR/Cas9-mediated mutagenesis confirms spr^tu21 is a mutation in the cyclin B1 gene. (A) Schematic representation of the CRISPR/Cas9 system, recognizing the target site within exon 2 of the cyclin B1 gene. Blue, guide RNA (sgRNA); green, target site where ag is the acceptor site of intron 1; red, PAM site. (B) Diagram of CRISPR/Cas9-mediated mutagenesis. Embryos from a wild-type cross (G₀) were injected at the 2-cell stage with the cas9 mRNA and sgRNA. After 3 months, founders were complementation tested to spr^tu21heterozygotes. In the G1, 10% of the eggs were spr^tu21/ro1transheterozygotes suggesting that the germline clone was approximately 40% of the germline. After outcrossing the G1 spr^ro1founder to wild-type fish, identification of the spr^ro1 carriers (G₂) was done by incrossing. (C–F) In vivo morphology and expression of cyclin B1 mRNA in: (C′) wild-type, (D′) spr^tu21, (E′) spr^tu21/ro1, and (F′) spr^ro1mutant embryos at 25 h. All embryos are shown in side view. Note that the spr^tu21/ro1 (E′), and spr^ro1 (F′) mutant embryos have zygotic cyclin B1mRNA transcripts at 20 h, but spr^tu21 mutant embryos do not. (G) Chromatograph sequence showing a splice-site mutation occurs in the spr^ro1. The spr^ro1 mutant has a 2-base deletion (AG acceptor site) and 4 base insertion (GGAT), underlined. Green, sgRNA target site; red, PAM site.