Recent applications of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system have greatly improved the efficiency of genome editing in pigs. However, in some cases, genetically modified pig models need an additional modification to improve their application. The objective of this experiment was to determine whether a combination of somatic cell NT (SCNT) by using a previously modified donor cell line and subsequent zygote injection with CRISPR/Cas9 guide RNA to target a second gene would result in embryos and offspring successfully containing both modifications. Fibroblast cell lines were collected from fumarylacetoacetate hydrolase deficient (FAH–/–) fetuses and used as the donor cell line. Somatic cell NT was performed by standard technique. A CRISPR guide RNA specific for recombination activating gene 2 (RAG2) was designed and in vitro transcribed from a synthesised gBlock (IDT) containing a T7 promoter sequence, the CRISPR Guide RNA (20 bp), and 85 bp of tracer RNA. The gBlock was PCR amplified with Q5 polymerase (NEB, Ipswich, MA, USA) and in vitro transcribed with the MEGAshortscript™ T7 Transcription Kit (Life Technologies, Grand Island, NY, USA). Guide RNA (20 ng μL–1) and polyadenylated Cas9 (20 ng μL–1, Sigma, St. Louis, MO, USA) were co-injected into the cytoplasm of SCNT zygotes at 14 to 16 h after fusion and activation. Injected SCNT were then cultured in vitro in PZM3 + 1.69 mM arginine medium (MU1) to Day 5. Three embryo transfers were performed surgically into recipient gilts on Day 4 or 5 of oestrus (50, 62, or 70 embryos per pig) to evaluate in vivo development. The remaining embryos were cultured in MU1 to Day 7 and analysed for the presence of modifications to the RAG2 gene. Embryos were classified as modified if they contained an INDEL as measured by both gel electrophoresis and DNA sequencing of PCR amplicons spanning the targeted exon. The RAG2 modification rate was 83.3% (n = 6), of which 50% (n = 3) of the embryos contained biallelic modifications. All control embryos contained a wild-type RAG2 gene (n = 5). Embryo transfer resulted in a 33.3% pregnancy rate (1/3). The combination of SCNT and CRISPR/Cas9 zygote injection can be a highly efficient tool to successfully create pig embryos with an additional modification. This additional technique further improves the usefulness of already created genetically modified pig models.
This study was funded by the National Institutes of Health via U42 OD011140.
Establishment of sperm cryopreservation and in vitro fertilisation protocols for rats
