CRISPR-Mediated Gene Editing in Mouse Embryos

Fertilized oocytes at the single-cell stage were harvested from FVB/N females on E0.5 and cultured in EmbryoMax^® Advanced KSOM Embryo Medium (EMD Millipore) at 37°C in an atmosphere containing 5% CO₂ before and after microinjection. Microinjections were performed as previously described^{11 (link)} and consisted of 25 ng/μl TrueGuide^™ sgRNA and 50 ng/μl TrueCut^™ Cas9 Protein v2 (Invitrogen) diluted in EmbryoMax^® Electroporation Buffer (EMD Millipore). The sgRNA targeting T/brachyury exon 3 was the same as in our earlier publication^{11 (link)}. A maximum of 28 injected zygotes were transferred bilaterally into the infundibulum of the uterine horns of pseudopregnant CD-1 females. Surrogate females were euthanized at E10.5, and conceptuses were recovered for morphological analysis and genotyping.