Generation of a Commercial-Scale Founder Population of Porcine Reproductive and Respiratory Syndrome Virus Resistant Pigs Using CRISPR-Cas.

Disease resistance genes in livestock provide health benefits to animals and opportunities for farmers to meet the growing demand for affordable, high-quality protein. Previously, researchers used gene editing to modify the porcine CD163 gene and demonstrated resistance to a harmful virus that causes porcine reproductive and respiratory syndrome (PRRS). To maximize potential benefits, this disease resistance trait needs to be present in commercially relevant breeding populations for multiplication and distribution of pigs. Toward this goal, a first-of-its-kind, scaled gene editing program was established to introduce a single modified CD163 allele into four genetically diverse, elite porcine lines. This effort produced healthy pigs that resisted PRRS virus infection as determined by macrophage and animal challenges. This founder population will be used for additional disease and trait testing, multiplication, and commercial distribution upon regulatory approval. Applying CRISPR-Cas to eliminate a viral disease represents a major step toward improving animal health.

Thrap3 promotes nonalcoholic fatty liver disease by suppressing AMPK-mediated autophagy.

Autophagy functions in cellular quality control and metabolic regulation. Dysregulation of autophagy is one of the major pathogenic factors contributing to the progression of nonalcoholic fatty liver disease (NAFLD). Autophagy is involved in the breakdown of intracellular lipids and the maintenance of healthy mitochondria in NAFLD. However, the mechanisms underlying autophagy dysregulation in NAFLD remain unclear. Here, we demonstrate that the hepatic expression level of Thrap3 was significantly increased in NAFLD conditions. Liver-specific Thrap3 knockout improved lipid accumulation and metabolic properties in a high-fat diet (HFD)-induced NAFLD model. Furthermore, Thrap3 deficiency enhanced autophagy and mitochondrial function. Interestingly, Thrap3 knockout increased the cytosolic translocation of AMPK from the nucleus and enhanced its activation through physical interaction. The translocation of AMPK was regulated by direct binding with AMPK and the C-terminal domain of Thrap3. Our results indicate a role for Thrap3 in NAFLD progression and suggest that Thrap3 is a potential target for NAFLD treatment.

Ultrasound-Guided Lumbar Intradiscal Injection: A Case Report With a High-Resolution Ultrasound Image.

We present a case report demonstrating a new technique for intradiscal injection under ultrasound guidance in treating lumbar disc herniation. A 16-year-old female gymnast underwent this procedure and experienced relief from pain. Traditional methods have been noted for their technical challenges and potential risk of nerve root damage. In this case, our approach visualized the lateral side of the disc and improved needle visibility. This technique potentially offers a clearer high-resolution confirmation of the needle's position within the disc. It is considered that this technique is effective not only for performing precise injections but also for enhancing safety due to the clear depiction of the needle tip entering the intervertebral disc.

Establishment, characterization, and validation of novel porcine embryonic fibroblasts as a potential source for genetic modification.

Fibroblasts are the common cell type in the connective tissue-the most abundant tissue type in the body. Fibroblasts are widely used for cell culture, for the generation of induced pluripotent stem cells (iPSCs), and as nuclear donors for somatic cell nuclear transfer (SCNT). We report for the first time, the derivation of embryonic fibroblasts (EFs) from porcine embryonic outgrowths, which share similarities in morphology, culture characteristics, molecular markers, and transcriptional profile to fetal fibroblasts (FFs). We demonstrated the efficient use of EFs as nuclear donors in SCNT, for enhanced post-blastocyst development, implantation, and pregnancy outcomes. We further validated EFs as a source for CRISPR/Cas genome editing with overall editing frequencies comparable to that of FFs. Taken together, we established an alternative and efficient pipeline for genome editing and for the generation of genetically engineered animals.

Effect of manipulation technique using ultrasound-guided cervical nerve root block on range of motion at the shoulder joint in frozen shoulder: a retrospective study.

PURPOSE: The aim of this study was to evaluate the range of motion (ROM) at the shoulder joint before and after silent manipulation. METHODS: This retrospective study included all patients who underwent silent manipulation at our institution between January 2013 and December 2017. In total, 1,665 shoulders in 1,610 patients (519 men, 1,146 women; mean age 55.4 ± 8.8 years) were treated during the study period. The mean symptom duration was 6.6 ± 7.1 months. ROM at the shoulder joint was measured in flexion, abduction, and external rotation before silent manipulation and at 1 week and 1, 2, and 3 months after the procedure. RESULTS: Mean ROM at the shoulder was 98.8° (95% confidence interval [CI] 97.9-99.8) before silent manipulation and 155.5° (154.1-156.8) after 3 months in flexion (p = 0.0000), 75.6° (74.5-76.8) and 152.9° (151.0-154.9), respectively, in abduction (p = 0.0000), and 12.7° (12.0-13.4) and 45.9° (44.4-47.4) in external rotation (p = 0.0000). All ROM values were significantly increased at all time points after the procedure. There were no unanticipated adverse events or serious adverse reactions. CONCLUSIONS: This study reports on the efficacy and safety of manipulation using conduction anesthesia for shoulder contractures in a large group of patients. Silent manipulation can increase ROM at the shoulder safely and effectively.

Genome-wide recombination map construction from single sperm sequencing in cattle.

BACKGROUND: Meiotic recombination is one of the important phenomena contributing to gamete genome diversity. However, except for human and a few model organisms, it is not well studied in livestock, including cattle. RESULTS: To investigate their distributions in the cattle sperm genome, we sequenced 143 single sperms from two Holstein bulls. We mapped meiotic recombination events at high resolution based on phased heterozygous single nucleotide polymorphism (SNP). In the absence of evolutionary selection pressure in fertilization and survival, recombination events in sperm are enriched near distal chromosomal ends, revealing that such a pattern is intrinsic to the molecular mechanism of meiosis. Furthermore, we further validated these findings in single sperms with results derived from sequencing its family trio of diploid genomes and our previous studies of recombination in cattle. CONCLUSIONS: To our knowledge, this is the first large-scale single sperm whole-genome sequencing effort in livestock, which provided useful information for future studies of recombination, genome instability, and male infertility.

Extraembryonic Endoderm (XEN) Cells Capable of Contributing to Embryonic Chimeras Established from Pig Embryos.

Most of our current knowledge regarding early lineage specification and embryo-derived stem cells comes from studies in rodent models. However, key gaps remain in our understanding of these developmental processes from nonrodent species. Here, we report the detailed characterization of pig extraembryonic endoderm (pXEN) cells, which can be reliably and reproducibly generated from primitive endoderm (PrE) of blastocyst. Highly expandable pXEN cells express canonical PrE markers and transcriptionally resemble rodent XENs. The pXEN cells contribute both to extraembryonic tissues including visceral yolk sac as well as embryonic gut when injected into host blastocysts, and generate live offspring when used as a nuclear donor in somatic cell nuclear transfer (SCNT). The pXEN cell lines provide a novel model for studying lineage segregation, as well as a source for genome editing in livestock.

Results of repeat manipulation under ultrasound-guided cervical nerve root block with corticosteroid and local anaesthetic injection for recurrence of frozen shoulder.

BACKGROUND: This study aimed to evaluate the clinical results of a repeat manipulation under ultrasound-guided cervical nerve root block (MUC) with corticosteroid and local anaesthetic injection for recurrence of idiopathic frozen shoulder after MUC. METHODS: A consecutive series of 42 shoulders in 39 patients with idiopathic frozen shoulder underwent MUC. All patients were assessed according to the American Shoulder Elbow Surgeon (ASES) score and shoulder range of motion (ROM) both before MUC and at 1 year thereafter. If patients continued to have pain and limited ROM at 3 months after the procedure, they were offered a repeat MUC. Such patients were also assessed before the procedure and at 3 months and 1 year thereafter. RESULTS: The initial MUC was successful in 31 shoulders (single group). Repeat MUC was required in 11 shoulders (repeat group). Patients in the single group showed significant improvement in ROM and ASES score at 1 year after the procedure (p < 0.001); similarly, patients in the repeat group had significant improvement in ROM and ASES score at 3 months and 1 year after the procedure (p < 0.001). Patients in the repeat group had had significantly more severely limited ROM (p < 0.01) and decreased ASES score (p < 0.001) before the procedure compared with those in the single group. CONCLUSIONS: A repeat MUC with corticosteroid and local anaesthetic injection is a valuable option before proceeding to surgery for recurrence of idiopathic frozen shoulder. When there is severely limited ROM and decreased ASES score before the MUC, a repeat MUC may be necessary, which would require the patient's informed consent. TRIAL REGISTRATION: Retrospectively registered.

One-Step Homology Mediated CRISPR-Cas Editing in Zygotes for Generating Genome Edited Cattle.

Selective breeding and genetic modification have been the cornerstone of animal agriculture. However, the current strategy of breeding animals over multiple generations to introgress novel alleles is not practical in addressing global challenges such as climate change, pandemics, and the predicted need to feed a population of 9 billion by 2050. Consequently, genome editing in zygotes to allow for seamless introgression of novel alleles is required, especially in cattle with long generation intervals. We report for the first time the use of CRISPR-Cas genome editors to introduce novel PRNP allelic variants that have been shown to provide resilience towards human prion pandemics. From one round of embryo injections, we have established six pregnancies and birth of seven edited offspring, with two founders showing >90% targeted homology-directed repair modifications. This study lays out the framework for in vitro optimization, unbiased deep-sequencing to identify editing outcomes, and generation of high frequency homology-directed repair-edited calves.

Donor-derived spermatogenesis following stem cell transplantation in sterile NANOS2 knockout males.

Spermatogonial stem cell transplantation (SSCT) is an experimental technique for transfer of germline between donor and recipient males that could be used as a tool for biomedical research, preservation of endangered species, and dissemination of desirable genetics in food animal populations. To fully realize these potentials, recipient males must be devoid of endogenous germline but possess normal testicular architecture and somatic cell function capable of supporting allogeneic donor stem cell engraftment and regeneration of spermatogenesis. Here we show that male mice, pigs, goats, and cattle harboring knockout alleles of the NANOS2 gene generated by CRISPR-Cas9 editing have testes that are germline ablated but otherwise structurally normal. In adult pigs and goats, SSCT with allogeneic donor stem cells led to sustained donor-derived spermatogenesis. With prepubertal mice, allogeneic SSCT resulted in attainment of natural fertility. Collectively, these advancements represent a major step toward realizing the enormous potential of surrogate sires as a tool for dissemination and regeneration of germplasm in all mammalian species.

Targeted Mutation of NGN3 Gene Disrupts Pancreatic Endocrine Cell Development in Pigs.

The domestic pig is an attractive model for biomedical research because of similarities in anatomy and physiology to humans. However, key gaps remain in our understanding of the role of developmental genes in pig, limiting its full potential. In this publication, the role of NEUROGENIN 3 (NGN3), a transcription factor involved in endocrine pancreas development has been investigated by CRISPR/Cas9 gene ablation. Precomplexed Cas9 ribonucleoproteins targeting NGN3 were injected into in vivo derived porcine embryos, and transferred into surrogate females. On day 60 of pregnancy, nine fetuses were collected for genotypic and phenotypic analysis. One of the piglets was identified as an in-frame biallelic knockout (Δ2/Δ2), which showed a loss of putative NGN3-downstream target genes: NEUROD1 and PAX4, as well as insulin, glucagon, somatostatin and pancreatic polypeptide-Y. Fibroblasts from this fetus were used in somatic cell nuclear transfer to generate clonal animals to qualify the effect of mutation on embryonic lethality. Three live piglets were born, received colostrum and suckled normally, but experienced extreme weight loss over a 24 to 36-hour period requiring humane euthanasia. Expression of pancreatic endocrine hormones: insulin, glucagon, and somatostatin were lost. The data support a critical role of NGN3 in porcine endocrine pancreas development.

Construction of PRDM9 allele-specific recombination maps in cattle using large-scale pedigree analysis and genome-wide single sperm genomics.

PRDM9 contributes to hybrid sterility and species evolution. However, its role is to be confirmed in cattle, a major domesticated livestock species. We previously found an association near PRDM9 with cattle recombination features, but the causative variants are still unknown. Using millions of genotyped cattle with pedigree information, we characterized five PRDM9 alleles and generated allele-specific recombination maps. By examining allele-specific recombination patterns, we observed the impact of PRDM9 on global distribution of recombination, especially in the two ends of chromosomes. We also showed strong associations between recombination hotspot regions and functional mutations within PRDM9 zinc finger domain. More importantly, we found one allele of PRDM9 to be very different from others in both protein composition and recombination landscape, indicating the causative role of this allele on the association between PRDM9 and cattle recombination. When comparing recombination maps from sperm and pedigree data, we observed similar genome-wide recombination patterns, validating the quality of pedigree-based results. Collectively, these evidence supported PRDM9 alleles as causal variants for the reported association with cattle recombination. Our study comprehensively surveyed the bovine PRDM9 alleles, generated allele-specific recombination maps, and expanded our understanding of the role of PRDM9 on genome distribution of recombination.

Genome editing and genetic engineering in livestock for advancing agricultural and biomedical applications.

Genetic modification of livestock has a longstanding and successful history, starting with domestication several thousand years ago. Modern animal breeding strategies predominantly based on marker-assisted and genomic selection, artificial insemination, and embryo transfer have led to significant improvement in the performance of domestic animals, and are the basis for regular supply of high quality animal derived food. However, the current strategy of breeding animals over multiple generations to introduce novel traits is not realistic in responding to the unprecedented challenges such as changing climate, pandemic diseases, and feeding an anticipated 3 billion increase in global population in the next three decades. Consequently, sophisticated genetic modifications that allow for seamless introgression of novel alleles or traits and introduction of precise modifications without affecting the overall genetic merit of the animal are required for addressing these pressing challenges. The requirement for precise modifications is especially important in the context of modeling human diseases for the development of therapeutic interventions. The animal science community envisions the genome editors as essential tools in addressing these critical priorities in agriculture and biomedicine, and for advancing livestock genetic engineering for agriculture, biomedical as well as "dual purpose" applications.

Targeted gene knock-in by CRISPR/Cas ribonucleoproteins in porcine zygotes.

The domestic pig is an important "dual purpose" animal model for agricultural and biomedical applications. There is an emerging consensus in the biomedical community for the use of large animal models such as pigs to either serve as an alternative, or complement investigations from the mouse. However, the use of pig has not proven popular due to technical difficulties and time required in generating models with desired genetic modifications. In this regard, the ability to directly modify the genome in the zygote and generate edited animals is highly desirable. This report demonstrates for the first time, the generation of gene targeted animals by direct injection of Cas9 ribonucleoprotein complex and short stretches of DNA sequences into porcine zygotes. The Cas9 protein from Streptococcus pyogenes was pre-complexed with a single guide RNA targeting downstream of the ubiquitously expressed COL1A gene, and co-injected with a single-stranded repair template into porcine zygotes. Using this approach a line of pigs that carry pseudo attP sites within the COL1A locus to enable phiC31 integrase mediated introduction of transgenes has been generated. This new route for genome engineering in pigs via zygote injection should greatly enhance applications in both agriculture and biomedicine.

Generation of germline ablated male pigs by CRISPR/Cas9 editing of the NANOS2 gene.

Genome editing tools have revolutionized the generation of genetically modified animals including livestock. In particular, the domestic pig is a proven model of human physiology and an agriculturally important species. In this study, we utilized the CRISPR/Cas9 system to edit the NANOS2 gene in pig embryos to generate offspring with mono-allelic and bi-allelic mutations. We found that NANOS2 knockout pigs phenocopy knockout mice with male specific germline ablation but other aspects of testicular development are normal. Moreover, male pigs with one intact NANOS2 allele and female knockout pigs are fertile. From an agriculture perspective, NANOS2 knockout male pigs are expected to serve as an ideal surrogate for transplantation of donor spermatogonial stem cells to expand the availability of gametes from genetically desirable sires.

Somatic Cell Nuclear Transfer Followed by CRIPSR/Cas9 Microinjection Results in Highly Efficient Genome Editing in Cloned Pigs.

The domestic pig is an ideal "dual purpose" animal model for agricultural and biomedical research. With the availability of genome editing tools such as clustered regularly interspaced short palindromic repeat (CRISPR) and associated nuclease Cas9 (CRISPR/Cas9), it is now possible to perform site-specific alterations with relative ease, and will likely help realize the potential of this valuable model. In this article, we investigated for the first time a combination of somatic cell nuclear transfer (SCNT) and direct injection of CRISPR/Cas ribonucleoprotein complex targeting GRB10 into the reconstituted oocytes to generate GRB10 ablated Ossabaw fetuses. This strategy resulted in highly efficient (100%) generation of biallelic modifications in cloned fetuses. By combining SCNT with CRISPR/Cas9 microinjection, genome edited animals can now be produced without the need to manage a founder herd, while simultaneously eliminating the need for laborious in vitro culture and screening. Our approach utilizes standard cloning techniques while simultaneously performing genome editing in the cloned zygotes of a large animal model for agriculture and biomedical applications.

Targeted Gene Knockin in Porcine Somatic Cells Using CRISPR/Cas Ribonucleoproteins.

The pig is an ideal large animal model for genetic engineering applications. A relatively short gestation interval and large litter size makes the pig a conducive model for generating and propagating genetic modifications. The domestic pig also shares close similarity in anatomy, physiology, size, and life expectancy, making it an ideal animal for modeling human diseases. Often, however, the technical difficulties in generating desired genetic modifications such as targeted knockin of short stretches of sequences or transgenes have impeded progress in this field. In this study, we have investigated and compared the relative efficiency of CRISPR/Cas ribonucleoproteins in engineering targeted knockin of pseudo attP sites downstream of a ubiquitously expressed COL1A gene in porcine somatic cells and generated live fetuses by somatic cell nuclear transfer (SCNT). By leveraging these knockin pseudo attP sites, we have demonstrated subsequent phiC31 integrase mediated integration of green fluorescent protein (GFP) transgene into the site. This work for the first time created an optimized protocol for CRISPR/Cas mediated knockin in porcine somatic cells, while simultaneously creating a stable platform for future transgene integration and generating transgenic animals.

Dynamic changes in nuclear import of a nuclear localisation signal-bearing substrate in 8-cell stage porcine embryos.

Coordinated intracellular trafficking is critically important for proper timing of major cellular events during embryogenesis. Nuclear import mediated by the karyopherin α/ß (importin α/ß) heterodimer is perhaps the best characterised nuclear trafficking system in eukaryotic cells. Seven karyopherin α subtypes have been identified in the domestic pig, and although each karyopherin α subtype transports proteins bearing classical nuclear localisation signals (NLSs), individual karyopherin α subtypes have been shown to preferentially transport specific cargoes. The aim of the present study was to determine the mechanism by which BRN2, a transcription factor previously reported to be transported by the karyopherin α/ß heterodimer, gains access to the nucleus in porcine oocytes and embryos. Using a combination of in vivo and in vitro assays, we tested the hypothesis that discrete karyopherin α subtypes transport BRN2 into the nuclei of porcine oocytes and cleavage stage embryos. Our results show that ectopically expressed BRN2 adopts a nuclear localisation in all nuclei through the 4-cell stage of development, whereas only a subset of blastomeres in 8-cell stage embryos possess nuclear BRN2. This pattern is unique to BRN2 because another ectopically expressed NLS-containing protein is able to adopt a nuclear localisation in all blastomeres of 8-cell stage embryos.

Characterization of pig sperm hyaluronidase and improvement of the digestibility of cumulus cell mass by recombinant pSPAM1 hyaluronidase in an in vitro fertilization assay.

Although sperm hyaluronidase is thought to play an important role in mammalian fertilization, the molecular function underlying these steps remains largely unknown. In mouse models, sperm-specific SPAM1 and HYAL5 hyaluronidase are believed to function in both sperm penetration of the cumulus matrix and sperm-ZP binding. However, gene-targeting studies for SPAM1 or HYAL5 show that hyaluronidases are not essential for fertilization, despite the fact that exogenous hyaluronidase can disrupt the cumulus matrix. Therefore, to evaluate whether sperm hyaluronidase is essential for mammalian fertilization, it is necessary to generate HYAL5/SPAM1 double-knockout mice. However, generating double-knockout mice is very difficult because these two genes exist on the same chromosome. Recently, investigators have begun to employ the pig model system to study human disease due to its similarities to human anatomy and physiology. In this study, we confirmed that pig SPAM1 exists as a single copy gene on chromosome 18 and is specifically expressed in the testis. In addition, we expressed recombinant pig SPAM1 in human embryonic kidney 293 cells and showed that these enzymes possess hyaluronidase activity. We also demonstrated that a polyclonal antibody against pig sperm hyaluronidase inhibits sperm-egg interactions in an in vitro fertilization (IVF) assay. Our results suggest that pig SPAM1 may play a critical role in pig fertilization and that recombinant SPAM1 can disperse the oocyte-cumulus complex in an IVF assay.

Utero-tubal embryo transfer and vasectomy in the mouse model.

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic alterations originated during preimplantation development on subsequent fetal development and adult health. The use of an effective and consistent embryo transfer technique is crucial to enhance the generation of genetically modified animals and to determine the effect of different treatments on implantation rates and survival to term. Embryos at the blastocyst stage are usually transferred by uterine transfer, performing a puncture in the uterine wall to introduce the embryo manipulation pipette. The orifice performed in the uterus does not close after the pipette has been withdrawn, and the embryos can outflow to the abdominal cavity due to the positive pressure of the uterus. The puncture can also produce a hemorrhage that impairs implantation, blocks the transfer pipette and may affect embryo development, especially when embryos without zona are transferred. Consequently, this technique often results in very variable and overall low embryo survival rates. Avoiding these negative effects, utero-tubal embryo transfer take advantage of the utero-tubal junction as a natural barrier that impedes embryo outflow and avoid the puncture of the uterine wall. Vasectomized males are required for obtaining pseudopregnant recipients. A technique to perform vasectomy is described as a complement to the utero-tubal embryo transfer.