Figure 2

Figure 2

Generation of positive and negative conditional allele pairs at the sox10 locus through the Bi-FoRe strategy. (A) Schematic diagram of the improved KI strategy based on the bidirectional multi-purpose Bi-FoRe donor consisting of two functional components. The Forward component (highlighted by red shadow) is designed to maintain the function of the sox10 gene, and the Reverse component (highlighted by green shadow) is designed to disrupt the sox10 function. The sox10 CRISPR/Cas target site is shown in dark blue, and the hEMX1 target site is shown in light blue, located in the middle of the two functional components in the donor, to facilitate unbiased identification of both forward and reverse integrations of the donor. (B) Z-stack confocal images of a 48 hpf sox10^{+/Bi−66-FoR-ReG−71} F₁ embryo from outcross of founder #3 and a 48 hpf sox10^{+/Bi−71-ReG-FoR−66−1} F₁ embryo from outcross of founder #1, respectively. Scale bar, 200 μm. (C) Junction PCR and direct sequencing results of F₁ progeny showing tdTomato expression from outcross of F₀ #3 or #4, demonstrating forward insertion of the sox10 Bi-FoRe donor. KI: pooled genomic DNA template of F₁ embryos from outcross of F₀ #3. WT: pooled genomic DNA template of wild-type embryos. (D) Junction PCR and direct sequencing results of F₁ progeny showing EGFP expression from outcross of F₀ #1 or #7, demonstrating reverse insertion of the sox10 Bi-FoRe donor. KI: pooled genomic DNA template of the F₁ embryos from outcross of F₀ #1. WT: pooled genomic DNA template of wild-type embryos. (E) Z-stack confocal images of a 56 hpf sox10^{Bi−66-FoR-ReG−71/Bi−71-ReG-FoR−66−2} F₁ embryo from the cross between founders #3 and #7, showing overlapping expression of tdTomato and EGFP. Scale bar, 200 μm