Fig. 5

Fig. 5 Establishment of 5’-end-modified dsDNA mediated KI system based on S-NGG-25 KI strategy.

a A schematic diagram of unmodified linearized dsDNA, 5’-modified linearized dsDNA, and circular dsDNA donors. All of the donors have the same integration cassette, including two homologous arms, a CDS, a linker, an eGFP coding sequence, and two polyA signals. 5’-modifications were added to the 5’-terminus of the linearized dsDNAs by using modified PCR primers. b, c Percentages of GFP-positive embryos after 1-cell-stage microinjection of Cas9/sgRNA together with different 5’-modified linearized dsDNA, unmodified linearized dsDNA, or circular dsDNA donors. cx43.4 and cx43 loci were chosen to evaluate modifications including PS, C6, C12, biotin, spacer18, and acrydite. GFP-positive embryos were counted at 48 hpf. At least 100 embryos were analyzed for each condition. Data represent mean ± SD of 3 independent experiments. P-values were calculated using an unpaired Student’s t-test. **P < 0.01; ***P < 0.001; ****P < 0.0001. d Germline transmission rates of tagged cx43.4 or cx43 using the 12PS-modified linearized donors. “G+” represents GFP-positive F₀, and “G−” represents GFP-negative F₀. At least 4 GFP-positive or 20 GFP-negative F₀ embryos were raised to adulthood and outbred with WT zebrafish. The number of F₀ that can generate the desired F₁ was counted to calculate the germline transmission rate. e, f Images of cx43.4^{+/+linear-eGFP} (e) and cx43^{+/+linear-eGFP} (f) F₁ embryos derived from outbreeding the desired F₀. Images were taken at 48 hpf. Scale bars, 100 µm. Images are representatives of at least 10 GFP-positive F₁ embryos.