Figure 4. Generation of geno-tagging at the sox10 locus and summary of our dual-cassette donor KI strategy.

(A) Z-stack confocal images of 48 hpf zebrafish embryos after the injection of the sox10 geno-tagging donor knockin system at the one-cell stage (Control) followed by the further injection of 25 pg Cre mRNA into a single cell at the 4 cell stage (+Cre mRNA). White arrowheads indicate the colocalization of the tdGFP and tdTomato signals. Scale bar, 100 μm. (B) Junction PCR and clonal sequencing to detect the knockin and Cre-induced recombination events in the injected embryos showing mosaic double-fluorescence signals. Injected: Embryos injected with the donor, Cas9/gRNA system and Cre mRNA. Donor: sox10 geno-tagging donor plasmid. Uninjected: Uninjected embryos. (C) Summary of the applications and potential expansion of our dual-cassette donor KI strategy.

Figure 4—figure supplement 1. Generation and evaluation of the sox10 geno-tagging allele.

(A) The position and sequence of the sox10 intron 3 (I3) target site designed for the Cas9/gRNA system. The protospacer sequence is shown in red, and the PAM is shown in green. (B) Targeting efficiency evaluated by PCR and AciI restriction endonuclease digestion. The result indicates that the indel efficiency is nearly 85%. (C) Sequencing results of the uncut PCR products (corresponding to indel mutations) from B after cloning. (D) The donor design and geno-tagging KI strategy at the sox10 locus. Primers S10qF and S10qR are used for qRT-PCR in G and H. (E) Phenotype analysis of the 48 hpf F₂ embryos from the incrossing of sox10^+/PoR-NeG heterozygotes (derived from #6 F₀) after the injection of Cre mRNA at the one-cell stage. The upper panel shows an uninjected control embryo bearing red fluorescent signals with normal pigmentation, whose genotype should be either sox10^+/PoR-NeG or sox10^{PoR-NeG/PoR-NeG}. The middle panel represents one Cre-injected embryo showing slightly less pigmentation but with only green fluorescent signals, indicating an efficient switch to the expression of tdGFP from that of tdTomoto after Cre injection; therefore, the genotype should be sox10^+/NeG. The lower panel shows a Cre-injected embryo devoid of body pigmentation that faithfully recapitulates the expected phenotype of the sox10 loss-of-function mutation. Similar to the previous embryo, this embryo shows only green fluorescent signals due to the Cre-induced efficient switch of the expression of the fluorescent reporter gene; therefore, the genotype is most likely tbx5a^NeG/NeG. The white arrowheads indicate otic vesicles, whose detailed structure can be seen under higher magnification of the boxed areas. Scale bar, 200 μm. (F) Genotyping results of the injected F₂ embryos in E determined via 5’ junction PCR analysis. Since all the defective embryos showed only green (tdGFP) and no red (tdTomoto) fluorescent signal, the PCR products are most likely derived from the amplification of the sox10^NeG allele. (G) qRT-PCR results showing the transcription level of the sox10 locus in wild-type (WT) and sox10 PoR-NeG geno-tagging donor KI zebrafish embryos at 72 hpf, using S10qF and S10qR primers. The sox10^+/NeG and sox10^+/PoR-NeG embryos were obtained from the crossing of sox10^{PoR-NeG/PoR-NeG} homozygotes with wild-type zebrafish with or without the injection of Cre mRNA, respectively. The average expression level of wild-type embryos was set as 1. (H) qRT-PCR results using S10qF and S10qR primers, showing the transcription level of the tbx5a locus in the sox10^+/NeG and sox10^NeG/NeG embryos derived from the Cre mRNA-injected sox10^+/PoR-NeG and sox10^{PoR-NeG/PoR-NeG} embryos, respectively. The original embryos were obtained from the crossing of sox10^{PoR-NeG/PoR-NeG} homozygotes with sox10^+/PoR-NeG heterozygote zebrafish. The expression levels in the KI embryos were normalized to the WT ones. Data are presented as the mean ±s.d., and a two-tailed Student’s t-test was applied to calculate p values in all the experiments. *: p<0.05. ***: p<0.001. NS: Not significant.