Cas9-green fluorescence protein (GFP) (cat no.10008100), crRNA, tracrRNA (cat no.1072533), and ATTO550-labeled tracrRNA (cat no. 1075928) were obtained from Integrated DNA Technologies Inc. (Coralville, IA, USA). crRNA was designed to target the tyrosinase gene of C57BL/6 mice (5′-GGGTGGATGACCGTGAGTCC-3′), which participates in melanin biosynthesis [10 (link)]. This gene is specifically expressed in retinal pigment epithelial cells of the eye, choroidal melanocytes, and hair follicle melanocytes in mammals [11 ]. It is possible to discriminate the results of genome editing from the eye color of offspring derived from C57BL/6 × ICR embryos without genetic analysis by knocking out the tyrosinase gene. The nuclease solution for embryo electroporation contained 200 ng/μl Cas9-GFP, 15 μM crRNA, 15 μM tracrRNA or a mixture solution with 7.5 μM tracrRNA and 7.5 μM tracrRNA-ATTO550 in Opti-MEM (Thermo Fisher Scientific Inc., MA, USA) [8 (link)] was prepared just before electroporation.
Protocol detail
CRISPR-Cas9 Mediated Tyrosinase Knockout
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Biosynthesis
C57bl mice
Choroidal
Color of eye
Crrna
Electroporation
Embryo
Epithelial cells
Gene
Genetic knocking out
Green fluorescence protein
Hair follicle
Mammals
Melanin
Melanocytes
Retinal pigment
Tracrrna
Tyrosinase
Corresponding Organization :
Other organizations : Iwate University, Integrated DNA Technologies (United States)
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Variable analysis
independent variables
- CrRNA design targeting the tyrosinase gene of C57BL/6 mice
- Cas9-GFP concentration
- CrRNA concentration
- TracrRNA concentration
- TracrRNA-ATTO550 concentration
- Genome editing results as indicated by eye color of offspring derived from C57BL/6 × ICR embryos
- Opti-MEM as the nuclease solution
- C57BL/6 × ICR embryos
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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