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 C

2

C

12

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.