Fig. 2

Evaluation of the S-25 strategy by tagging all zebrafish connexins.

a Percentages of GFP-positive F₀ embryos after tagging cx30.3, cx34.4, cx35, cx39.9, cx43, cx43.4, cx44.1, cx47.1, cx48.5, cx52.6, or cx55.5. S-25 strategy was performed and GFP-positive F₀ embryos were counted at 48 hpf. At least 200 embryos were analyzed for each gene. b Number of the desired F₀ for cx30.3, cx34.4, cx35, cx44.1, cx47.1, cx48.5, cx52.6, or cx55.5 KI. Genomic DNAs were extracted from the caudal fins of 96 injected F₀ zebrafish (except for cx55.5, the genomic DNAs of which were from 72 F₀ zebrafish) and used for 5’-junction PCR. PCR-positive F₀ are those positive for 5’-junction PCR. Screened F₀ are the PCR-positive F₀ outbred with WT zebrafish. Desired F₀ are the screened F₀ whose F₁ offspring had precisely repaired 5’-junction. Two high-efficiency sgRNAs were chosen for each gene KI (sg-1 and sg-2). c Correlation between the cleavage efficiencies of sgRNAs and the ratios of PCR-positive F₀. The cleavage efficiency of a sgRNA was evaluated by PCR product enzymatic digestions at 24-h post injection or software analysis by the TIDE website. d Germline transmission rates of PCR-positive F₀ and PCR-negative F₀ for cx30.3 and cx34.4 KI. “P+”, PCR-positive F₀; “P-”, PCR-negative F₀. Two high-efficiency sgRNAs were used for KI at either locus. At least 6 F₀ were analyzed for each group. e Mosaicism of the germline of F₀ founders for cx30.3 and cx34.4 KI was determined by the percentage of F₁ carrying the KI cassette. PCR-positive cx30.3^+/+eGFP or cx34.4^+/+eGFP F₁ were shown in blue and PCR-negative cx30.3^+/+ or cx34.4^+/+ F₁ were in orange. More than 70 F₁ were examined for each PCR-positive F₀ founder.