Design of the donor DNA templates for homologous recombination

For precise gene replacement using homologous recombination in the fcy1 gene, donor DNA templates (with a 25-bp deletion of the fcy1sg2 sequence) with homology arms of 1 kb, 0.5 kb, or 0.2 kb, were constructed using overlap extension PCR. Approximately 1-kb genomic fragments containing 5’-upstream and 3’-downstream sequences were amplified using CI50/CI51 and CI52/CI53, respectively (Fig. 1a). These fragments were fused by CI50/CI53 to obtain the intact donor DNA, followed by amplification with TB11/TB12 and TB9/TB10 to reduce the length of homologous arms to 0.5 kb and 0.2 kb, respectively (Fig. 1b). The resulting fragments were designated as donor DNA with homology arms of 1 kb (2071 bp in total), 0.5 kb (1008 bp in total), and 0.2 kb (407 bp in total), respectively (Additional file 1: Table S2).

Targeted gene replacement using homologous recombination along with CRISPR/Cas9. a A schematic diagram of the procedure used to construct the donor DNA template that lacks the sgRNA sequence for homologous recombination. b A schematic diagram showing the method for fcy1 mutation using CRISPR/Cas9 and donor DNA. PCR fragments with around 1 kb, 0.5 kb, and 0.2 kb each of right and left homologous arms were used as donor DNA temples. Black arrows display the primer pairs used for the PCR experiments in Additional file 1: Fig. S2C