RecOFAR facilitate dual nicks-based efficient gene KI at multiple loci in zebrafish. (A) Schematic overview of the strategy to generate an Ndr2-linker-dendra2-V5 KI allele. The nested PCR primers used for knock-in identification are shown (Supplementary Table S4). (B) Germline transmission rate of linker-dendra2-V5 precise integration at the Ndr2 locus with double-nicking approach. All four Rec factors are required for the enhancing effect. (C) Germline transmission rate of linker-dendra2 precise integration at the Ndr2 locus with DSB approach. (D) Dendra2 fluorescence pattern in the Ndr2-linker-dendra2-V5 KI-positive F2 embryos. Scale bar, 100 μm. (E) Schematic overview of Lefty2 target KI and the germline transmission rate of linker-dendra2 precise integration at the Lefty2 locus. The nested PCR primers used for knock-in identification are shown (Supplementary Table S4). (F) Representative Southern blotting analysis of the Lefty2 targeted allele. T, KI target band. WT, wild type band. (G) Dendra2 fluorescence pattern in the Lefty2-linker-dendra2-V5 KI-positive F2 embryos. Scale bar, 100 μm. (H) Schematic overview of the strategy to generate a Bmp2b-linker-dendra2-V5-linker allele. The nested PCR primers used for knock-in identification are shown (Supplementary Table S4). (I) Germline transmission rate of linker-Dendra2 precise integration at the Bmp2b loci with different strategies. (J) Dendra2 fluorescence pattern in the Bmp2b-linker-Dendra2-V5-linker KI-positive F2 embryos. Scale bar, 100 μm. (K) Schematic overview of the strategy to generate the GFAP-5.5kb KI allele. The nested PCR primers used for knock-in identification are shown (Supplementary Table S4). (L) Germline transmission rate of 5.5 kb precise integration at the gfap locus.
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