Cas9-expressing chickens and pigs as resources for genome editing in livestock

Genetically modified animals continue to provide important insights into the molecular basis of health and disease. Research has focused mostly on genetically modified mice, although other species like pigs resemble the human physiology more closely. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR-Cas9. Here, we report the generation of transgenic chickens and pigs that constitutively express Cas9 in all organs. These animals are healthy and fertile. Functionality of Cas9 was confirmed in both species for a number of different target genes, for a variety of cell types and in vivo by targeted gene disruption in lymphocytes and the developing brain, and by precise excision of a 12.7-kb DNA fragment in the heart. The Cas9 transgenic animals will provide a powerful resource for in vivo genome editing for both agricultural and translational biomedical research, and will facilitate reverse genetics as well as cross-species comparisons

Porcine model elucidates function of p53 isoform in carcinogenesis and reveals novel circTP53 RNA

Recent years have seen an increasing number of genetically engineered pig models of human diseases including cancer. We previously generated pigs with a modified TP53 allele that carries a Cre-removable transcriptional stop signal in intron 1, and an oncogenic mutation TP53R167H (orthologous to human TP53R175H) in exon 5. Pigs with the unrecombined mutant allele (flTP53R167H) develop mainly osteosarcoma but also nephroblastomas and lymphomas. This observation suggested that TP53 gene dysfunction is itself the key initiator of bone tumorigenesis, but raises the question which aspects of the TP53 regulation lead to the development of such a narrow tumour spectrum. Molecular analysis of p53 revealed the presence of two internal TP53 promoters (Pint and P2) equivalent to those found in human. Consequently, both pig and human express TP53 isoforms. Data presented here strongly suggest that P2-driven expression of the mutant R167H-Δ152p53 isoform (equivalent to the human R175H-Δ160p53 isoform) and its circular counterpart circTP53 determine the tumour spectrum and play a critical role in the malignant transformation in flTP53R167H pigs. The detection of Δ152p53 isoform mRNA in serum is indicative of tumorigenesis. Furthermore, we showed a tissue-specific p53-dependent deregulation of the p63 and p73 isoforms in these tumours. This study highlights important species-specific differences in the transcriptional regulation of TP53. Considering the similarities of TP53 regulation between pig and human, these observations provide useful pointers for further investigation into isoform function including the novel circTP53 in both the pig model and human patients.ISSN:0950-9232ISSN:1476-559

Maternal placenta modulates a deleterious fetal mutation

Intrauterine growth restriction (IUGR) is caused by dysregulation of placental metabolism. Paternally inherited IUGR mutations in the fetus influence maternal physiology via the placenta. However, it is not known whether the maternal placenta also affects the extent of IUGR in such fetuses. In cattle and other ruminants, maternal-fetal communication occurs primarily at the placentomes. We previously identified a 3 deletion in the noncoding MER1 repeat containing imprinted transcript 1 (MIMT1) gene that, when inherited from the sire, causes IUGR and late abortion in Ayshire cattle with variable levels of severity. Here, we compared the transcriptome and genomic imprinting in fetal and maternal placentome components of wild-type and MIMT1(Del/WT) fetuses before IUGR became apparent, to identify key early events. Transcriptome analysis revealed fewer differentially expressed genes in maternal than fetal MIMT1(Del/WT) placentome. AST1, within the PEG3 domain, was the only gene consistently reduced in IUGR in both fetal and maternal samples. Several genes showed an imprinting pattern associated with IUGR, of which only secernin 3 (SCRN3) and paternally expressed 3 (PEG3) were differentially imprinted in both placentome components. Loss of strictly monoallelic, allele-specific expression (similar to 80:20) of PEG3 in the maternal MIMT1(Del/WT) placenta could be associated with incomplete penetrance of MIMT1(Del). Our data show that dysregulation of the PEG3 domain is involved in IUGR, but also reveal that maternal placental tissues may affect the penetrance of the paternally inherited IUGR mutation.Peer reviewe

Efficient production of multi-modified pigs for xenotransplantation by ‘combineering’, gene stacking and gene editing

Xenotransplantation from pigs could alleviate the shortage of human tissues and organs for transplantation. Means have been identified to overcome hyperacute rejection and acute vascular rejection mechanisms mounted by the recipient. The challenge is to combine multiple genetic modifications to enable normal animal breeding and meet the demand for transplants. We used two methods to colocate xenoprotective transgenes at one locus, sequential targeted transgene placement - ‘gene stacking’, and cointegration of multiple engineered large vectors - ‘combineering’, to generate pigs carrying modifications considered necessary to inhibit short to mid-term xenograft rejection. Pigs were generated by serial nuclear transfer and analysed at intermediate stages. Human complement inhibitors CD46, CD55 and CD59 were abundantly expressed in all tissues examined, human HO1 and human A20 were widely expressed. ZFN or CRISPR/Cas9 mediated homozygous GGTA1 and CMAH knockout abolished α-Gal and Neu5Gc epitopes. Cells from multi-transgenic piglets showed complete protection against human complement-mediated lysis, even before GGTA1 knockout. Blockade of endothelial activation reduced TNFα-induced E-selectin expression, IFNγ-induced MHC class-II upregulation and TNFα/cycloheximide caspase induction. Microbial analysis found no PERV-C, PCMV or 13 other infectious agents. These animals are a major advance towards clinical porcine xenotransplantation and demonstrate that livestock engineering has come of age

Viable pigs after simultaneous inactivation of porcine MHC class I and three xenoreactive antigen genes GGTA1, CMAH and B4GALNT2.

BACKGROUND Cell surface carbohydrate antigens play a major role in the rejection of porcine xenografts. The most important for human recipients are α-1,3 Gal (Galactose-alpha-1,3-galactose) causing hyperacute rejection, also Neu5Gc (N-glycolylneuraminic acid) and Sd(a) blood group antigens both of which are likely to elicit acute vascular rejection given the known human immune status. Porcine cells with knockouts of the three genes responsible, GGTA1, CMAH and B4GALNT2, revealed minimal xenoreactive antibody binding after incubation with human serum. However, human leucocyte antigen (HLA) antibodies cross-reacted with swine leucocyte antigen class I (SLA-I). We previously demonstrated efficient generation of pigs with multiple xeno-transgenes placed at a single genomic locus. Here we wished to assess whether key xenoreactive antigen genes can be simultaneously inactivated and if combination with the multi-transgenic background further reduces antibody deposition and complement activation. METHODS Multiplex CRISPR/Cas9 gene editing and somatic cell nuclear transfer were used to generate pigs carrying functional knockouts of GGTA1, CMAH, B4GALNT2 and SLA class I. Fibroblasts derived from one- to four-fold knockout animals, and from multi-transgenic cells (human CD46, CD55, CD59, HO1 and A20) with the four-fold knockout were used to examine the effects on human IgG and IgM binding or complement activation in vitro. RESULTS Pigs were generated carrying four-fold knockouts of important xenoreactive genes. In vitro assays revealed that combination of all four gene knockouts reduced human IgG and IgM binding to porcine kidney cells more effectively than single or double knockouts. The multi-transgenic background combined with GGTA1 knockout alone reduced C3b/c and C4b/c complement activation to such an extent that further knockouts had no significant additional effect. CONCLUSION We showed that pigs carrying several xenoprotective transgenes and knockouts of xenoreactive antigens can be readily generated and these modifications will have significant effects on xenograft survival