Precise PA-tag Integration in Mice

To insert three tandem copies of PA-tag sequences just upstream from the stop codon (Fig. 1b), 3×PA-tag sequences flanked by 129- and 118-bp homology arms were artificially synthesized (Integrated DNA Technologies). To avoid unwanted excision of PA-tag sequences by the internal recombination among three copies, synonymous substitutions were introduced into each tag sequences. By using this synthesized DNA as the template, long single-stranded DNA (ssDNA) donors were prepared with the Phospho-PCR method (Guide-it Long ssDNA Production System, Takara Bio; Inoue et al., 2021 (link)). The mixture of annealed crRNA/tracrRNA, Cas9 protein, and long ssDNA donor was injected into mouse zygotes at the concentrations of 38, 50, and 25 ng/μl, and the zygotes were transferred into the oviduct of surrogate mothers to raise pups.