dCas9-fusion plasmids, in vitro transcription, zygote microinjections and embryo culture

The dCas9-Zfp57[KRAB]-T2A-GFP fusion construct was generated using the pLV hUbC-dCas9-VP64-T2A-GFP plasmid backbone (30). The NheI sites were used to remove VP64 and introduce mouse ZFP57 (amino acids 13–55 encoding the KRAB domain). We also generated the control plasmid, dCas9-control, with no insert (Nhe1 self-ligation). In vitro transcription (IVT) was utilized to generate full-length RNA for zygote injections using the mMESSAGE mMACHINE^® T7 Ultra Kit (Thermofisher). The IVT template was the PCR product generated using the dCas9-Zfp57[KRAB]-T2A-GFP construct amplified with a forward primer encompassing the T7 RNA polymerase promoter upstream of the dCas9 sequence and a reverse primer with a stop codon and polyA tail designed to the end of the GFP (primers detailed in Supplementary Table S1). The in vitro transcription, DNAseI treatment and LiCl clean-up were performed following the suppliers recommendations. The resulting IVT RNA was resuspended in TE and quantified by spectrophotometer (Nanodrop). To ensure IVT products were full-length without degradation, an aliquot of IVT RNA was visualized following gel electrophoresis on a 1.5% agarose gels prepared under MOPS buffer/formaldehyde denaturing conditions. Embryos were collected from 5 to 7 week-old F₁ (C57BL/6J × CBA/H) super-ovulated females crossed with F₁ males. Superovulation was induced by intraperitoneal injection of pregnant mare serum gonadotropin (PMSG, Intervet, 5 IU) and human chorionic gonadotropin (hCG, Intervet, 7.5 IU) 46–48 h later. Early zygotes were collected at 18h hCG injection and microinjected with 1–2pl of 200 ng/μl dCas9-Zfp57[KRAB]-T2A-GFP mRNA, 20 ng/μl of crRNA and 20 ng/μl of tracrRNA. Embryos were cultured in K-modified simplex optimized medium microdrops under oil at 37°C, 5% CO₂ for 78 h until the blastocyst stage.