Mammalian melanin synthesis is dependent on the oxidation of tyrosine. Tyrosinase (Tyr) is a key enzyme that catalyzes the oxidation of tyrosine. When exogenous Tyr
is integrated into the genome of white coat color mouse, it can render the mouse to obtain the function of melanin synthesis, presenting a different coat color. To rapidly generate the mouse model with Tyr
gene integrated, in this study, we constructed a promotorless plasmid donor pTyr-2A-DsRed, which containis the coding sequences of Tyr
gene and the red fluorescent reporter (DsRed), and the two flanking homologous arms. We selected the first intron of Rosa26
for targeting integration, and designed ZFN pair, TALEN pairs and CRISPR/Cas9 cutting at almost the same site. Through measuring the intensity of DsRed fluorescence in C2
cells by flow cytometry, we compared the efficiency of the targeted integration of exogenous DNA mediated by the three different genome editing tools, and found that CRISPR/Cas9 was the most efficient. Therefore, we further integrated the plasmid donor into the genome of mouse embryonic stem (ES) cells, and screening the targeted single cell clones for blastocyst injection and subsequent embryo transfer. Ultimately, a single survived chimeric mouse with plasmid donor integrated was obtained. This mouse presented a white color hair mixed with black color hair phenotype, indicating that the targeted integration of Tyr
gene in Rosa26
locus was correctly expressed.