Fig. 6

Fig. 6

a Timeline. Cre mRNA injections into progeny of 3C tyr animals at the 1-cell stage elicit ubiquitous recombination (blue box). A subset of injected embryos was heat treated at 12 or 60 hpf to trigger Cas9-GFP expression and mutagenesis of the tyr target site (green box). In addition, Cre mRNA injected animals were collected at 120 hpf without a prior heat treatment. Single embryo genotyping was applied to all specimen and ten embryos of each time point (heat: none; heat: 12 hpf; heat 60 hpf) were combined and analyzed using next generation sequencing (NGS). In addition, siblings of all three time points were raised to adulthood (6 months postfertilization (mpf)). b Animals subjected to Cre mRNA injection but no heat treatment show neither a pigmentation phenotype at embryonic or adult stages nor any signs of mutagenesis. c A strong pigmentation phenotype is observed at embryonic (50 hpf) and adult stages (6 mpf) in animals heat treated at 12 hpf. (Note almost complete absence of pigmentation in the RPE at 6 mpf making the eye appear red.) NGS confirms high-level indel production. d Pigmentation defects are observed at adult but not at embryonic stages in animals heat treated at 60 hpf. Sequencing corroborates mutagenesis of the tyr target site. Larval examples shown are representatives across three experiments showing the same result. Adult examples shown are representative of >15 individual fish examined. e The recombined 3C tyr allele is transmitted in the germline of F0 animals subjected to Cre mRNA injection only. Heat treatment in the F1 generation results in GFP expression and pigmentation defects after activation at 12 hpf. Examples shown are representatives across ten experiments showing the same result. A total of >300 GFP-positive individuals and their respective non-GFP-positive siblings were analyzed. Scale bars: 500 µm for 50 hpf and 2 mm for 6 mpf in b–d; 150 µm in e, except for lower row with 500 µm.