Superovulated female C57BL/6 mice were mated to B6D2F1 males. A cocktail solution containing 50 ng/µl gRNA and 100 ng/µl Cas9 mRNA was injected into one blastomere of two-cell embryos so that resulting embryos would be mosaic for the Fzd2 mutation with at least 50% of the cells being wild type. Embryos were incubated at 37°C, 5% CO2 in KSOM medium. The KSOM culture drops were covered with mineral oil to prevent evaporation. After cocktail injection, the two-cell embryos were transferred the same day into the oviducts of Swiss Webster E0.5 pseudopregnant recipient females, which were synchronized by using Swiss Webster vasectomized males. Micromanipulation, embryo collection and embryo transfers were performed at room temperature in HEPES-buffered CZB medium. Founder mice were crossed with wild-type C57BL/6 mice to yield Fzd2INS/+ offspring. Fzd2INS/+ male and female mice were intercrossed to produce Fzd2INS/+, Fzd2INS/INS and Fzd2+/+ mice for analysis.