In vitro genome editing activity of Cas9 in somatic cells after random and transposon-based genomic Cas9 integration.

Due to its close resemblance, the domesticated pig has proven to be a diverse animal model for biomedical research and genome editing tools have contributed to developing porcine models for several human diseases. By employing the CRISPR-Cas9 system, porcine embryos or somatic cells can be genetically modified to generate the desired genotype. However, somatic cell nuclear transfer (SCNT) of modified somatic cells and embryo manipulation are challenging, especially if the desired genotype is detrimental to the embryo. Direct in vivo edits may facilitate the production of genetically engineered pigs by integrating Cas9 into the porcine genome. Cas9 expressing cells were generated by either random integration or transposon-based integration of Cas9 and used as donor cells in SCNT. In total, 15 animals were generated that carried a transposon-based Cas9 integration and two pigs a randomly integrated Cas9. Cas9 expression was confirmed in muscle, tonsil, spleen, kidney, lymph nodes, oral mucosa, and liver in two boars. Overall, Cas9 expression was higher for transposon-based integration, except in tonsils and liver. To verify Cas9 activity, fibroblasts were subjected to in vitro genome editing. Isolated fibroblasts were transfected with guide RNAs (gRNA) targeting different genes (GGTA1, B4GALNT2, B2M) relevant to xenotransplantation. Next generation sequencing revealed that the editing efficiencies varied (2-60%) between the different target genes. These results show that the integrated Cas9 remained functional, and that Cas9 expressing pigs may be used to induce desired genomic modifications to model human diseases or further evaluate in vivo gene therapy approaches.

Generation and first characterization of TRDC-knockout pigs lacking γδ T cells.

The TRDC-locus encodes the T cell receptor delta constant region, one component of the γδ T cell receptor which is essential for development of γδ T cells. In contrast to peptide recognition by αß T cells, antigens activating γδ T cells are mostly MHC independent and not well characterized. Therefore, the function of γδ T cells and their contribution to protection against infections is still unclear. Higher numbers of circulating γδ T cells compared to mice, render the pig a suitable animal model to study γδ T cells. Knocking-out the porcine TRDC-locus by intracytoplasmic microinjection and somatic cell nuclear transfer resulted in healthy living γδ T cell deficient offspring. Flow cytometric analysis revealed that TRDC-KO pigs lack γδ T cells in peripheral blood mononuclear cells (PBMC) and spleen cells. The composition of the remaining leucocyte subpopulations was not affected by the depletion of γδ T cells. Genome-wide transcriptome analyses in PBMC revealed a pattern of changes reflecting the impairment of known or expected γδ T cell dependent pathways. Histopathology did not reveal developmental abnormalities of secondary lymphoid tissues. However, in a vaccination experiment the KO pigs stayed healthy but had a significantly lower neutralizing antibody titer as the syngenic controls.

CRISPR/Cas12a mediated knock-in of the Polled Celtic variant to produce a polled genotype in dairy cattle.

In modern livestock farming horned cattle pose an increased risk of injury for each other as well as for the farmers. Dehorning without anesthesia is associated with stress and pain for the calves and raises concerns regarding animal welfare. Naturally occurring structural variants causing polledness are known for most beef cattle but are rare within the dairy cattle population. The most common structural variant in beef cattle consists of a 202 base pair insertion-deletion (Polled Celtic variant). For the generation of polled offspring from a horned Holstein-Friesian bull, we isolated the Polled Celtic variant from the genome of an Angus cow and integrated it into the genome of fibroblasts taken from the horned bull using the CRISPR/Cas12a system (formerly Cpf1). Modified fibroblasts served as donor cells for somatic cell nuclear transfer and reconstructed embryos were transferred into synchronized recipients. One resulting pregnancy was terminated on day 90 of gestation for the examination of the fetus. Macroscopic and histological analyses proved a polled phenotype. The remaining pregnancy was carried to term and delivered one calf with a polled phenotype which died shortly after birth. In conclusion, we successfully demonstrated the practical application of CRISPR/Cas12a in farm animal breeding and husbandry.

Possible detrimental effects of beta-2-microglobulin knockout in pigs.

BACKGROUND: Despite major improvements in pig-to-primate xenotransplantation, long-term survival of xenografts is still challenging. The major histocompatibility complex (MHC) class I, which is crucial in cellular immune response, is an important xenoantigen. Abrogating MHC class I expression on xenografts might be beneficial for extending graft survival beyond current limits. METHODS: In this study, we employed the CRISPR/Cas9 system to target exon 2 of the porcine beta-2-microglobulin (B2M) gene to abrogate SLA class I expression on porcine cells. B2M-KO cells served as donor cells for somatic cell nuclear transfer, and cloned embryos were transferred to three recipient sows. The offspring were genotyped for mutations at the B2M locus, and blood samples were analyzed via flow cytometry for the absence of SLA class I molecules. RESULTS: Pregnancies were successfully established and led to the birth of seven viable piglets. Genomic sequencing proved that all piglets carried biallelic modifications at the B2M locus leading to a frameshift, a premature stop codon, and ultimately a functional knockout. However, survival times of these animals did not exceed 4 weeks due to unexpected disease processes. CONCLUSION: Here, we demonstrate the feasibility of generating SLA class I knockout pigs by targeting the porcine beta-2-microglobulin gene using the CRISPR/Cas9 system. Additionally, our findings indicate for the first time that this genetic modification might have a negative impact on the viability of the animals. These issues need to be solved to unveil the real value for xenotransplantation in the future.

Efficient production of biallelic GGTA1 knockout pigs by cytoplasmic microinjection of CRISPR/Cas9 into zygotes.

BACKGROUND: Xenotransplantation is considered to be a promising solution to the growing demand for suitable donor organs for transplantation. Despite tremendous progress in the generation of pigs with multiple genetic modifications thought to be necessary to overcoming the severe rejection responses after pig-to-non-human primate xenotransplantation, the production of knockout pigs by somatic cell nuclear transfer (SCNT) is still an inefficient process. Producing genetically modified pigs by intracytoplasmic microinjection of porcine zygotes is an alluring alternative. The porcine GGTA1 gene encodes for the α1,3-galactosyltransferase that synthesizes the Gal epitopes on porcine cells which constitute the major antigen in a xenotransplantation setting. GGTA1-KO pigs have successfully been produced by transfecting somatic cells with zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or CRISPR/Cas targeting GGTA1, followed by SCNT. METHODS: Here, we microinjected a CRISPR/Cas9 vector coding for a single-guide RNA (sgRNA) targeting exon 8 of the GGTA1 gene into the cytoplasm of 97 in vivo-derived porcine zygotes and transferred 86 of the microinjected embryos into three hormonally synchronized recipients. Fetuses and piglets were analyzed by flow cytometry for remaining Gal epitopes. DNA was sequenced to detect mutations at the GGTA1 locus. RESULTS: Two of the recipients remained pregnant as determined by ultrasound scanning on day 25 of gestation. One pregnancy was terminated on day 26, and six healthy fetuses were recovered. The second pregnancy was allowed to go to term and resulted in the birth of six healthy piglets. Flow cytometry analysis revealed the absence of Gal epitopes in four of six fetuses (66%), indicating a biallelic KO of GGTA1. Additionally, three of the six live-born piglets (50%) did not express Gal epitopes on their cell surface. Two fetuses and two piglets showed a mosaicism with a mixed population of Gal-free and Gal-expressing cells. Only a single piglet did not have any genomic modifications. Genomic sequencing revealed indel formation at the GGTA1 locus ranging from +17 bp to -20 bp. CONCLUSIONS: These results demonstrate the efficacy of CRISPR/Cas to generate genetic modifications in pigs by simplified technology, such as intracytoplasmic microinjection into zygotes, which would significantly facilitate the production of genetically modified pigs suitable for xenotransplantation. Importantly, this simplified injection protocol avoids the penetration of the vulnerable pronuclear membrane, and is thus compatible with higher survival rates of microinjected embryos, which in turn facilitates production of genetically modified piglets.

Sperm sex-sorting in the Asian elephant (Elephas maximus).

In captive Asian elephants, there is a strong need for production of female offspring to enhance reproduction, counter premature aging processes in female animals and reduce challenging management situations derived from husbandry of several bulls in one institution. Artificial insemination of flow cytometrically sex-sorted spermatozoa offers the possibility to predetermine the sex of offspring with high accuracy. The aims of this study were to determine a suitable semen extender and basic parameters for flow cytometrical sex-sorting of Asian elephant spermatozoa. In total 18 semen samples were collected by manual rectal stimulation from one bull. Sperm quality parameters and sex sortability of spermatozoa were evaluated after dilution in three semen extenders (MES-HEPES-skim milk, MES-HEPES, TRIS-citric acid) and DNA staining. MES-HEPES-skim milk was the only semen extender found suitable to sex Asian elephant spermatozoa. From 18 ejaculates collected, 12 were successfully sorted with a purity of 94.5+/-0.7% at an average sort rate of 1945.5+/-187.5 spermatozoa per second. Sperm integrity, progressive and total motility were 42.6+/-3.9%, 48.1+/-3.3%, 59.4+/-3.8% after DNA labelling, and 64.8+/-3.2%, 58.0+/-5.0%, 70.8+/-4.4% after sorting, respectively. After liquid storage of sorted spermatozoa for 12h at 4 degrees C, sperm integrity, progressive and total motility were 46.4+/-5.2%, 32.2+/-4.2% and 58.2+/-3.9%, respectively. The obtained results provide a promising base to inseminate Asian elephants with sexed semen.