Fig. 3

Reducing non-homologous residues introduced by lamGolden sequence increases the accuracy of MMEJ-mediated KI.

a A schematic diagram of S-NGG-25 and S-CCN-25 donors for cx43.4 KI. Sense or antisense lamGolden site is represented by “NGG” or “CCN”. Predicted cleavage sites of Cas9 were indicated by green dotted lines, and residuals left in homologous arms were shown in gray. b Percentages of GFP-positive embryos obtained by using S-CCN-25 or S-NGG-25 strategy for cx43.4 or cx43 KI. GFP-positive embryos were counted at 48 hpf. At least 100 embryos were analyzed for each strategy for each gene. Data represent mean ± SD of 3 independent experiments. P-values were calculated using an unpaired Student’s t-test. ns, not significant. c Numbers of desired F₀ obtained using S-CCN-25 or S-NGG-25 strategy. Heritable F₀ are the GFP-positive F₀ whose KI alleles can be detected in their F₁. Desired F₀ are the GFP-positive F₀ whose KI alleles were verified in their F₁ to be heritable and precise by 5’-junction PCR. d Images of F₀ embryos after tbx5a or tnni1b KI. Cas9 mRNA, sgRNA (targeting tbx5a or tnni1b), and the corresponding S-NGG-25 donor were co-injected into WT one-cell-stage embryos; images were taken at 4 and 7 days post fertilization (dpf). Deficient embryos were indicated by red arrowheads. WT embryos were used as the control. Scale bars, 1 mm. e, f Images of tbx5a^+/+eGFP and tnni1b^+/+eGFP F₁. White arrowheads indicate GFP signals in the heart and pectoral fin of a tbx5a^+/+eGFP larva at 48 hpf (e) and in the heart of a tnni1b^+/+eGFP larva at 48 hpf (f). Scale bars, 100 µm. Images are representatives of at least 10 larvae.