FIGURE 1.

Design of composite tag knock-in through the CRISPR-Cas9 system. In this design, an ∼200-nt length composite that contains either a FLAG or PA epitope tag in its trimeric form followed by a tobacco etch virus (TEV) protease cleavage site, a biotin acceptor domain (Bio tag), and a C-terminus HiBiT peptide tag was knocked into the 3′ end of the coding sequence of the sox3 gene. A long ssDNA donor fragment that contains the composite tag flanked at both ends by the homology arms that corresponds to the CDS upstream from the stop codon (5′ homology arm) and the 3′ UTR downstream from the stop codon (3′ homology arm) of the sox3 gene was used as a template to induce the homology-directed repair (HDR) mechanism after the CRISPR-Cas9-mediated double-strand break (DSB).