Fig. 1

S-25 donor ensures efficient MMEJ-mediated KI in zebrafish.

a Schematic diagrams of MMEJ-, HR-, and NHEJ-mediated KI strategies, respectively. MMEJ-based donor vector containing a single artificial consensus Cas9/sgRNA binding site (lamGolden) was called MMEJ-single (A1), while the one with two lamGolden sites was named MMEJ-double (A2). One-kb homologous arms were used in the design for HR-based donors (A3). The NHEJ-based donor was shown in A4. Exons, lamGolden sites, linkers, eGFP, and SV40+bGH polyA are colored blue, gray, orange, green, and blank, respectively. Introns are shown as black lines. Homologous arms are shown in yellow. Start and stop codons are labeled as green and red vertical lines, respectively. b, c Percentages of GFP-positive embryos for cx43.4 and cx43 KI, respectively. Each donor was co-microinjected into wild-type (WT) zebrafish embryos at the one-cell stage with the Cas9/sgRNA system. GFP-positive embryos were counted at 48 h post fertilization (hpf). Donors are as follows: S-10, S-25, S-40, and S-100 donors are MMEJ-single with 10-bp, 25-bp, 40-bp, and 100-bp homologous arms, respectively; D-10, D-25, D-40, and D-100 donors are MMEJ-double with 10-bp, 25-bp, 40-bp, and 100-bp homologous arms, respectively; +sgR, microinjection with lamGolden sgRNA; -sgR, microinjection without lamGolden sgRNA. At least 100 embryos were analyzed for each group in one experiment. Data represent mean ± SD of 3 independent experiments. P-values were calculated using an unpaired Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. d, f Images of F₁ zebrafish carrying KI alleles in cx43.4 or cx43 loci. White arrowheads indicate GFP signals in the notochord, spinal cord, cerebral cortex, cornea, and hatching gland of a cx43.4^+/+eGFP larva at 48 hpf (d) and in the notochord, spinal cord, lens capsule, and ultimobranchial body of a cx43^+/+eGFP larva at 48 hpf (f). Scale bars, 100 µm. Images are representatives of at least 10 larvae. e Germline transmission rates of GFP-tagged cx43.4 or cx43 using different strategies. “G+” represents GFP-positive F₀ zebrafish, and “G−” represents GFP-negative F₀ zebrafish. At least 6 F₀ were analyzed for each group. g Mosaicism of the germline of F₀ founders was determined by the percentage of F₁ carrying the KI cassette. GFP-positive cx43.4^+/+eGFP or cx43^+/+eGFP F₁ were in blue, and GFP-negative cx43.4^+/+ or cx43^+/+ F₁ were in orange. More than 100 F₁ were examined for each F₀ founder.