In Vitro Transcription of Cas9 mRNA and sgRNA.

sgRNAs were designed using the CRISPRtool (crispr.mit.edu) to minimize potential off-target effects. Sequences of sgRNA targeting exon 2 were as follows: GCAGCTGCATAAGTCGCCACC. T7 promoter was added to Cas9 by PCR amplification using primer. T7–Cas9 PCR product was gel purified and used as the template for in vitro transcription (IVT) using mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies). T7 promoter was added to sgRNAs template by PCR amplification The T7–sgRNA PCR product was gel purified and used as the template for IVT using MEGAshortscript T7 kit (Life Technologies). Both the Cas9 mRNA and the sgRNAs were purified using MEGAclear kit (Life Technologies) and eluted in RNase-free water. Cas mRNA (100 ng/μL) and sgRNA (50 ng/μL) were mixed and injected into zygotes at the pronuclei stage. The injected zygotes were cultured in KSOM with amino acids at 37 °C under 5% CO₂ in air until blastocyst stage by 3.5 d. Thereafter, 15–25 blastocysts were transferred into uterus of pseudopregnant imprinting control region females at 2.5 days postcoitum (dpc). Tail clips were subjected to a standard DNA extraction procedure (47). Briefly, a 2- to 3-mm mouse tail segment was lysed with 100 μL alkaline lysis reagent (25 mM NaOH, 0.2 mM EDTA in water, pH = 12), and the sample was heated with agitation at 95 °C for 30 min. A total of 100 μL neutralization buffer (40 mM Tris⋅HCl in water, pH = 5) was then added, and the sample was subjected to centrifugation at 10,000 × g for 5 min. A total of 1–2 μL supernatant was used for PCR genotyping.