Gene editing of pigs to control influenza A virus infections.

Proteolytic activation of the hemagglutinin (HA) glycoprotein by host cellular proteases is pivotal for influenza A virus (IAV) infectivity. Highly pathogenic avian influenza viruses possess the multibasic cleavage site of the HA which is cleaved by ubiquitous proteases, such as furin; in contrast, the monobasic HA motif is recognized and activated by trypsin-like proteases, such as the transmembrane serine protease 2 (TMPRSS2). Here, we aimed to determine the effects of TMPRSS2 on the replication of pandemic H1N1 and H3N2 subtype IAVs in the natural host, the pig. The use of the CRISPR/Cas 9 system led to the establishment of homozygous gene edited (GE) TMPRSS2 knockout (KO) pigs. Delayed IAV replication was demonstrated in primary respiratory cells of KO pigs in vitro. IAV infection in vivo resulted in significant reduction of virus shedding in the upper respiratory tract, and lower virus titers and pathological lesions in the lower respiratory tract of TMPRSS2 KO pigs as compared to WT pigs. Our findings could support the commercial use of GE pigs to minimize (i) the economic losses caused by IAV infection in pigs, and (ii) the emergence of novel IAVs with pandemic potential through genetic reassortment in the "mixing vessel", the pig.

Profiling development of abdominal organs in the pig.

The pig is an ideal model system for studying human development and disease due to its similarities to human anatomy, physiology, size, and genome. Further, advances in CRISPR gene editing have made genetically engineered pigs viable models for the study of human pathologies and congenital anomalies. However, a detailed atlas illustrating pig development is necessary for identifying and modeling developmental defects. Here we describe normal development of the pig abdominal system and show examples of congenital defects that can arise in CRISPR gene edited SAP130 mutant pigs. Normal pigs at different gestational ages from day 20 (D20) to term were examined and the configuration of the abdominal organs was studied using 3D histological reconstructions with episcopic confocal microscopy, magnetic resonance imaging (MRI) and necropsy. This revealed prominent mesonephros, a transient embryonic organ present only during embryogenesis, at D20, while the developing metanephros that will form the permanent kidney are noted at D26. By D64 the mesonephroi are absent and only the metanephroi remain. The formation of the liver and pancreas was observed by D20 and complete by D30 and D35 respectively. The spleen and adrenal glands are first identified at D26 and completed by D42. The developing bowel and the gonads are identified at D20. The bowel appears completely rotated by D42, and testes in the male were descended at D64. This atlas and the methods used are excellent tools for identifying developmental pathologies of the abdominal organs in the pig at different stages of development.

Improvements in pig agriculture through gene editing.

Genetic modification of animals via selective breeding is the basis for modern agriculture. The current breeding paradigm however has limitations, chief among them is the requirement for the beneficial trait to exist within the population. Desirable alleles in geographically isolated breeds, or breeds selected for a different conformation and commercial application, and more importantly animals from different genera or species cannot be introgressed into the population via selective breeding. Additionally, linkage disequilibrium results in low heritability and necessitates breeding over successive generations to fix a beneficial trait within a population. Given the need to sustainably improve animal production to feed an anticipated 9 billion global population by 2030 against a backdrop of infectious diseases and a looming threat from climate change, there is a pressing need for responsive, precise, and agile breeding strategies. The availability of genome editing tools that allow for the introduction of precise genetic modification at a single nucleotide resolution, while also facilitating large transgene integration in the target population, offers a solution. Concordant with the developments in genomic sequencing approaches, progress among germline editing efforts is expected to reach feverish pace. The current manuscript reviews past and current developments in germline engineering in pigs, and the many advantages they confer for advancing animal agriculture.

Chloride channel accessory 1 gene deficiency causes selective loss of mucus production in a new pig model.

Morbidity and mortality of respiratory diseases are linked to airway obstruction by mucus but there are still no specific, safe, and effective drugs to correct this phenotype. The need for better treatment requires a new understanding of the basis for mucus production. In that regard, studies of human airway epithelial cells in primary culture show that a mucin granule constituent known as chloride channel accessory 1 (CLCA1) is required for inducible expression of the inflammatory mucin MUC5AC in response to potent type 2 cytokines. However, it remained uncertain whether CLCLA1 is necessary for mucus production in vivo. Conventional approaches to functional biology using targeted gene knockout were difficult due to the functional redundancy of additional Clca genes in mice not found in humans. We reasoned that CLCA1 function might be better addressed in pigs that maintain the same four-member CLCA gene locus and the corresponding mucosal and submucosal populations of mucous cells found in humans. Here we develop to our knowledge the first CLCA1-gene-deficient (CLCA1-/-) pig and show that these animals exhibit loss of MUC5AC+ mucous cells throughout the airway mucosa of the lung without affecting comparable cells in the tracheal mucosa or MUC5B+ mucous cells in submucosal glands. Similarly, CLCA1-/- pigs exhibit loss of MUC5AC+ mucous cells in the intestinal mucosa without affecting MUC2+ mucous cells. These data establish CLCA1 function for controlling MUC5AC expression as a marker of mucus production and provide a new animal model to study mucus production at respiratory and intestinal sites.

Disruption of anthrax toxin receptor 1 in pigs leads to a rare disease phenotype and protection from senecavirus A infection.

Senecavirus A (SVA) is a cause of vesicular disease in pigs, and infection rates are rising within the swine industry. Recently, anthrax toxin receptor 1 (ANTXR1) was revealed as the receptor for SVA in human cells. Herein, the role of ANTXR1 as a receptor for SVA in pigs was investigated by CRISPR/Cas9 genome editing. Strikingly, ANTXR1 knockout (KO) pigs exhibited features consistent with the rare disease, GAPO syndrome, in humans. Fibroblasts from wild type (WT) pigs supported replication of SVA; whereas, fibroblasts from KO pigs were resistant to infection. During an SVA challenge, clinical symptoms, including vesicular lesions, and circulating viremia were present in infected WT pigs but were absent in KO pigs. Additional ANTXR1-edited piglets were generated that were homozygous for an in-frame (IF) mutation. While IF pigs presented a GAPO phenotype similar to the KO pigs, fibroblasts showed mild infection, and circulating SVA nucleic acid was decreased in IF compared to WT pigs. Thus, this new ANTXR1 mutation resulted in decreased permissiveness of SVA in pigs. Overall, genetic disruption of ANTXR1 in pigs provides a unique model for GAPO syndrome and prevents circulating SVA infection and clinical symptoms, confirming that ANTXR1 acts as a receptor for the virus.

Disrupting porcine glutaminase does not block preimplantation development and elongation nor decrease mTORC1 activation in conceptuses†.

Elongation of pig conceptuses is a dynamic process, requiring adequate nutrient provisions. Glutamine is used as an energy substrate and is involved in the activation of mechanistic target of rapamycin complex 1 (mTORC1) during porcine preimplantation development. However, the roles of glutamine have not been extensively studied past the blastocyst stage. Therefore, the objective of the current study was to determine if glutaminase (GLS), which is the rate-limiting enzyme in glutamine metabolism, was necessary for conceptus elongation to proceed and was involved in mTORC1 activation. The CRISPR/Cas9 system was used to induce loss-of-function mutations in the GLS gene of porcine fetal fibroblasts. Wild type (GLS+/+) and knockout (GLS-/-) fibroblasts were used as donor cells for somatic cell nuclear transfer, and GLS+/+ and GLS-/- blastocyst-stage embryos were transferred into surrogates. On day 14 of gestation, GLS+/+ conceptuses primarily demonstrated filamentous morphologies, and GLS-/- conceptuses exhibited spherical, ovoid, tubular, and filamentous morphologies. Thus, GLS-/- embryos were able to elongate despite the absence of GLS protein and minimal enzyme activity. Furthermore, spherical GLS-/- conceptuses had increased abundance of transcripts related to glutamine and glutamate metabolism and transport compared to filamentous conceptuses of either genotype. Differences in phosphorylation of mTORC1 components and targets were not detected regarding conceptus genotype or morphology, but abundance of two transcriptional targets of mTORC1, cyclin D1, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha was increased in spherical conceptuses. Therefore, porcine GLS is not essential for conceptus elongation and is not required for mTORC1 activation at this developmental timepoint.

Cardiovascular Development and Congenital Heart Disease Modeling in the Pig.

Background Modeling cardiovascular diseases in mice has provided invaluable insights into the cause of congenital heart disease. However, the small size of the mouse heart has precluded translational studies. Given current high-efficiency gene editing, congenital heart disease modeling in other species is possible. The pig is advantageous given its cardiac anatomy, physiology, and size are similar to human infants. We profiled pig cardiovascular development and generated genetically edited pigs with congenital heart defects. Methods and Results Pig conceptuses and fetuses were collected spanning 7 stages (day 20 to birth at day 115), with at least 3 embryos analyzed per stage. A combination of magnetic resonance imaging and 3-dimensional histological reconstructions with episcopic confocal microscopy were conducted. Gross dissections were performed in late-stage or term fetuses by using sequential segmental analysis of the atrial, ventricular, and arterial segments. At day 20, the heart has looped, forming a common atria and ventricle and an undivided outflow tract. Cardiac morphogenesis progressed rapidly, with atrial and outflow septation evident by day 26 and ventricular septation completed by day 30. The outflow and atrioventricular cushions seen at day 20 undergo remodeling to form mature valves, a process continuing beyond day 42. Genetically edited pigs generated with mutation in chromatin modifier SAP130 exhibited tricuspid dysplasia, with tricuspid atresia associated with early embryonic lethality. Conclusions The major events in pig cardiac morphogenesis are largely complete by day 30. The developmental profile is similar to human and mouse, indicating gene edited pigs may provide new opportunities for preclinical studies focused on outcome improvements for congenital heart disease.

Lack of airway submucosal glands impairs respiratory host defenses.

Submucosal glands (SMGs) are a prominent structure that lines human cartilaginous airways. Although it has been assumed that SMGs contribute to respiratory defense, that hypothesis has gone without a direct test. Therefore, we studied pigs, which have lungs like humans, and disrupted the gene for ectodysplasin (EDA-KO), which initiates SMG development. EDA-KO pigs lacked SMGs throughout the airways. Their airway surface liquid had a reduced ability to kill bacteria, consistent with SMG production of antimicrobials. In wild-type pigs, SMGs secrete mucus that emerges onto the airway surface as strands. Lack of SMGs and mucus strands disrupted mucociliary transport in EDA-KO pigs. Consequently, EDA-KO pigs failed to eradicate a bacterial challenge in lung regions normally populated by SMGs. These in vivo and ex vivo results indicate that SMGs are required for normal antimicrobial activity and mucociliary transport, two key host defenses that protect the lung.

The use of cells from ANPEP knockout pigs to evaluate the role of aminopeptidase N (APN) as a receptor for porcine deltacoronavirus (PDCoV).

The coronaviruses, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV) represent important sources of neonatal diarrhea on pig farms. The requirement for aminopeptidase N (APN) as a receptor for TGEV, but not for PEDV, is well established. In this study, the biological relevance of APN as a receptor for PDCoV was tested by using CRISPR/Cas9 to knockout the APN gene, ANPEP, in pigs. Porcine alveolar macrophages (PAMs) from ANPEP knockout (KO) pigs showed resistance to PDCoV infection. However, lung fibroblast-like cells, derived from the ANPEP KO PAM cultures, supported PDCoV infection to high levels. The results suggest that APN is a receptor for PDCoV in PAMs but is not necessary for infection of lung-derived fibroblast cells. The infection of the ANPEP KO pigs with PDCoV further confirmed that APN is dispensable as a receptor for PDCoV.

New perspective on conceptus estrogens in maternal recognition and pregnancy establishment in the pig†.

The proposed signal for maternal recognition of pregnancy in pigs is estrogen (E2), produced by the elongating conceptuses between days 11 to 12 of pregnancy with a more sustained increase during conceptus attachment and placental development on days 15 to 30. To understand the role of E2 in porcine conceptus elongation and pregnancy establishment, a loss-of-function study was conducted by editing aromatase (CYP19A1) using CRISPR/Cas9 technology. Wild-type (CYP19A1+/+) and (CYP19A1-/-) fibroblast cells were used to create embryos through somatic cell nuclear transfer, which were transferred into recipient gilts. Elongated and attaching conceptuses were recovered from gilts containing CYP19A1+/+ or CYP19A1-/- embryos on day 14 and 17 of pregnancy. Total E2 in the uterine flushings of gilts with CYP19A1-/- embryos was lower than recipients containing CYP19A1+/+ embryos with no difference in testosterone, PGF2α, or PGE2 on either day 14 or 17. Despite the loss of conceptus E2 production, CYP19A1-/- conceptuses were capable of maintaining the corpora lutea. However, gilts gestating CYP19A1-/- embryos aborted between days 27 and 31 of gestation. Attempts to rescue the pregnancy of CYP19A1-/- gestating gilts with exogenous E2 failed to maintain pregnancy. However, CYP19A1-/- embryos could be rescued when co-transferred with embryos derived by in vitro fertilization. Endometrial transcriptome analysis revealed that ablation of conceptus E2 resulted in disruption of a number biological pathways. Results demonstrate that intrinsic E2 conceptus production is not essential for pre-implantation development, conceptus elongation, and early CL maintenance, but is essential for maintenance of pregnancy beyond 30 days .

Transcriptional Regulation of Thrombospondins and Its Functional Validation through CRISPR/Cas9 Mediated Gene Editing in Corpus Luteum of Water Buffalo (Bubalus Bubalis).

BACKGROUND/AIMS: Thrombospondins (TSPs) are large multi-modular proteins, identified as natural angiogenesis inhibitors that exert their activity by binding to CD36 and CD47 receptors. The anti-angiogenic effect of TSPs in luteal regression of water buffalo has not been addressed. The present study characterized the expression pattern and localization of TSPs and their receptors in ovarian corpus luteum during different stages of development in buffalo. This study also elucidated the effect of exogenous Thrombospondin1 (TSP1) or the knocking out of the endogenous protein on luteal cell viability and function. Further, the in vitro transcriptional interaction of TSP1 with hormones, LH, PGF2α and angiogenic growth factors, VEGF and FGF2 were also evaluated. METHODS: First, the CLs were classified into four groups based on macroscopic observation and progesterone concentration. mRNA expression of examined factors was measured by qPCR, localization by immunoblotting and immunohistochemistry. TSP1 was knocked out (KO) in cultured luteal cells isolated from late luteal stage CLs (day 1116) by CRISPR/Cas9 mediated gene editing technology in order to functionally validate the TSP1 gene. Isolated cells from late stage CLs were also stimulated with different doses of TSP1, LH, PGF2α, VEGF and FGF2 for various time intervals to determine transcriptional regulation of thrombospondins. RESULTS: mRNA expression of TSPs and their receptors were found to be significantly higher in late and regressed stage of CL as compared to other groups which was consistent with the findings of immunoblotting and immunolocalization experiments. It was observed that TSP1 induced apoptosis, down regulated angiogenic growth factors, VEGF and FGF2 and attenuated progesterone production in cultured luteal cells. However, knocking out of endogenous TSP1 with CRISPR/Cas9 system improved the viability of luteal cells, progesterone synthesis and upregulated the expression of VEGF and FGF2 in the KO luteal cells. PGF2α induced the upregulation of TSPs and Caspase 3 transcripts, whereas treatment with LH and angiogenic growth factors (VEGF and FGF2) down regulated the TSP system in luteal cells. CONCLUSION: Collectively, these data provide evidence that thrombospondins along with their receptors are expressed at varying levels in different stages of CL progression with maximum expression during the late and regressing stages. These results are consistent with the hypothesis that thrombospondins stimulated by PGF2α plays an essential modulatory role in bringing about structural and functional luteolysis in buffalo.

Improvement of in vitro and early in utero porcine clone development after somatic donor cells are cultured under hypoxia.

Genetically engineered pigs serve as excellent biomedical and agricultural models. To date, the most reliable way to generate genetically engineered pigs is via somatic cell nuclear transfer (SCNT), however, the efficiency of cloning in pigs is low (1-3%). Somatic cells such as fibroblasts frequently used in nuclear transfer utilize the tricarboxylic acid cycle and mitochondrial oxidative phosphorylation for efficient energy production. The metabolism of somatic cells contrasts with cells within the early embryo, which predominately use glycolysis. We hypothesized that fibroblast cells could become blastomere-like if mitochondrial oxidative phosphorylation was inhibited by hypoxia and that this would result in improved in vitro embryonic development after SCNT. In a previous study, we demonstrated that fibroblasts cultured under hypoxic conditions had changes in gene expression consistent with increased glycolytic/gluconeogenic metabolism. The goal of this pilot study was to determine if subsequent in vitro embryo development is impacted by cloning porcine embryonic fibroblasts cultured in hypoxia. Here we demonstrate that in vitro measures such as early cleavage, blastocyst development, and blastocyst cell number are improved (4.4%, 5.5%, and 17.6 cells, respectively) when donor cells are cultured in hypoxia before nuclear transfer. Survival probability was increased in clones from hypoxic cultured donors compared to controls (8.5 vs. 4.0 ± 0.2). These results suggest that the clones from donor cells cultured in hypoxia are more developmentally competent and this may be due to improved nuclear reprogramming during somatic cell nuclear transfer.

Resistance to coronavirus infection in amino peptidase N-deficient pigs.

The alphacoronaviruses, transmissible gastroenteritis virus (TGEV) and Porcine epidemic diarrhea virus (PEDV) are sources of high morbidity and mortality in neonatal pigs, a consequence of dehydration caused by the infection and necrosis of enterocytes. The biological relevance of amino peptidase N (ANPEP) as a putative receptor for TGEV and PEDV in pigs was evaluated by using CRISPR/Cas9 to edit exon 2 of ANPEP resulting in a premature stop codon. Knockout pigs possessing the null ANPEP phenotype and age matched wild type pigs were challenged with either PEDV or TGEV. Fecal swabs were collected daily from each animal beginning 1 day prior to challenge with PEDV until the termination of the study. The presence of virus nucleic acid was determined by PCR. ANPEP null pigs did not support infection with TGEV, but retained susceptibility to infection with PEDV. Immunohistochemistry confirmed the presence of PEDV reactivity and absence of TGEV reactivity in the enterocytes lining the ileum in ANPEP null pigs. The different receptor requirements for TGEV and PEDV have important implications in the development of new genetic tools for the control of enteric disease in pigs.

Zygote injection of RNA encoding Cre recombinase results in efficient removal of LoxP flanked neomycin cassettes in pigs.

Genetically engineered pigs are often created with a targeting vector that contains a loxP flanked selectable marker like neomycin. The Cre-loxP recombinase system can be used to remove the selectable marker gene from the resulting offspring or cell line. Here is described a new method to remove a loxP flanked neomycin cassette by direct zygote injection of an mRNA encoding Cre recombinase. The optimal concentration of mRNA was determined to be 10 ng/µL when compared to 2 and 100 ng/µL (P < 0.0001). Development to the blastocyst stage was 14.1% after zygote injection with 10 ng/µL. This method successfully removed the neomycin cassette in 81.9% of injected in vitro derived embryos; which was significantly higher than the control (P < 0.0001). Embryo transfer resulted in the birth of one live piglet with a Cre deleted neomycin cassette. The new method described can be used to efficiently remove selectable markers in genetically engineered animals without the need for long term cell culture and subsequent somatic cell nuclear transfer.

Knockout of maternal CD163 protects fetuses from infection with porcine reproductive and respiratory syndrome virus (PRRSV).

After infection of the porcine dam at about 90 days of gestation, porcine reproductive and respiratory syndrome virus (PRRSV) crosses the placenta and begins to infect fetuses. Outcomes of include abortion, fetal death and respiratory disease in newborn piglets. CD163 is the receptor for the virus. In this study, CD163-positive fetuses, recovered between 109 days of gestation or 20 days after maternal infection, were completely protected from PRRSV in dams possessing a complete knockout of the CD163 receptor. The results demonstrate a practical means to eliminate PRRSV-associated reproductive disease, a major source of economic hardship to agriculture.

Gene editing as applied to prevention of reproductive porcine reproductive and respiratory syndrome.

Porcine Reproductive and Respiratory Syndrome (PRRS) causes severe reproductive failure in sows as well as transplacental transfer of PRRS virus (PRRSV) to late-gestation fetuses, resulting in abortions, early farrowing, increased number of stillborn piglets, and weak neonatal piglets. PRRSV-infected boars present with anorexia and lethargy, and have decreased sperm quality. The gene for the cellular receptor that the PRRSV uses, Cluster of differentiation 163 (CD163), was edited using Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene-editing technology to create biallelic DNA edits to the CD163 gene in 100% of the offspring. CD163-null pigs challenged with virus were completely resistant to both Type 1 and Type 2 PRRSV isolates, as measured by clinical signs, viremia, antibody response, and lung histopathology. In vitro studies showed that CD163-null alveolar macrophages were also not permissive to infection by a panel of six Type 1 and nine Type 2 viral isolates. Thus, DNA editing of the CD163 gene prevented PRRSV infection and reproductive losses associated with infection.

Genetic engineering alveolar macrophages for host resistance to PRRSV.

Standard strategies for control of porcine reproductive and respiratory syndrome virus (PRRSV) have not been effective, as vaccines have not reduced the prevalence of disease and many producers depopulate after an outbreak. Another method of control would be to prevent the virus from infecting the pig. The virus was thought to infect alveolar macrophages by interaction with a variety of cell surface molecules. One popular model had PRRSV first interacting with heparin sulfate followed by binding to sialoadhesin and then being internalized into an endosome. Within the endosome, PRRSV was thought to interact with CD163 to uncoat the virus so the viral genome could be released into the cytosol and infect the cell. Other candidate receptors have included vimentin, CD151 and CD209. By using genetic engineering, it is possible to test the importance of individual entry mediators by knocking them out. Pigs engineered by knockout of sialoadhesin were still susceptible to infection, while CD163 knockout resulted in pigs that were resistant to infection. Genetic engineering is not only a valuable tool to determine the role of specific proteins in infection by PRRSV (in this case), but also provides a means to create animals resistant to disease. Genetic engineering of alveolar macrophages can also illuminate the role of other proteins in response to infection. We suggest that strategies to prevent infection be pursued to reduce the reservoir of virus.

Replacement of Porcine CD163 Scavenger Receptor Cysteine-Rich Domain 5 with a CD163-Like Homolog Confers Resistance of Pigs to Genotype 1 but Not Genotype 2 Porcine Reproductive and Respiratory Syndrome Virus.

CD163 knockout (KO) pigs are resistant to infection with genotype 2 (type 2) porcine reproductive and respiratory syndrome virus (PRRSV). Furthermore, the substitution of CD163 scavenger receptor cysteine-rich (SRCR) domain 5 with a homolog of human CD163-like (hCD163L1) SRCR 8 domain confers resistance of transfected HEK cells to type 1 PRRSV. As a means to understand the role of domain 5 in PRRSV infection with both type 1 and type 2 viruses, pigs were genetically modified (GM) to possess one of the following genotypes: complete knockout (KO) of CD163, deletions within SRCR domain 5, or replacement (domain swap) of SRCR domain 5 with a synthesized exon encoding a homolog of hCD163L1 SRCR domain 8. Immunophenotyping of porcine alveolar macrophages (PAMs) showed that pigs with the KO or SRCR domain 5 deletion did not express CD163. When placed in culture, PAMs from pigs with the CD163 KO phenotype were completely resistant to a panel consisting of six type 1 and nine type 2 isolates. PAMs from pigs that possessed the hCD163L1 domain 8 homolog expressed CD163 and supported the replication of all type 2 isolates, but no type 1 viruses. Infection of CD163-modified pigs with representative type 1 and type 2 viruses confirmed the in vitro results. The results confirm that CD163 is the likely receptor for all PRRS viruses. Even though type 1 and type 2 viruses are considered phenotypically similar at several levels, there is a distinct difference between the viral genotypes in the recognition of CD163. IMPORTANCE: Genetic modification of the CD163 gene creates the opportunity to develop production animals that are resistant to PRRS, the costliest viral disease to ever face the swine industry. The results create further opportunities to develop refinements in the modification of CD163 with the goal of making pigs refractory to infection while retaining important CD163 functions.

Zygote injection of CRISPR/Cas9 RNA successfully modifies the target gene without delaying blastocyst development or altering the sex ratio in pigs.

The CRISPR/Cas9 genome editing tool has increased the efficiency of creating genetically modified pigs for use as biomedical or agricultural models. The objectives were to determine if DNA editing resulted in a delay in development to the blastocyst stage or in a skewing of the sex ratio. Six DNA templates (gBlocks) that were designed to express guide RNAs that target the transmembrane protease, serine S1, member 2 (TMPRSS2) gene were in vitro transcribed. Pairs of CRISPR guide RNAs that flanked the start codon and polyadenylated Cas9 were co-injected into the cytoplasm of zygotes and cultured in vitro to the blastocyst stage. Blastocysts were collected as they formed on days 5, 6 or 7. PCR was performed to determine genotype and sex of each embryo. Separately, embryos were surgically transferred into recipient gilts on day 4 of estrus. The rate of blastocyst development was not significantly different between CRISPR injection embryos or the non-injected controls at day 5, 6 or 7 (p = 0.36, 0.09, 0.63, respectively). Injection of three CRISPR sets of guides resulted in a detectable INDEL in 92-100 % of the embryos analyzed. There was not a difference in the number of edits or sex ratio of male to female embryos when compared between days 5, 6 and 7 to the controls (p > 0.22, >0.85). There were 12 resulting piglets and all 12 had biallelic edits of TMRPSS2. Zygote injection with CRISPR/Cas9 continues to be a highly efficient tool to genetically modify pig embryos.

Glycine supplementation in vitro enhances porcine preimplantation embryo cell number and decreases apoptosis but does not lead to live births.

Most in vitro culture conditions are less-than-optimal for embryo development. Here, we used a transcriptional-profiling database to identify culture-induced differences in gene expression in porcine blastocysts compared to in vivo-produced counterparts. Genes involved in glycine transport (SLC6A9), glycine metabolism (GLDC, GCSH, DLD, and AMT), and serine metabolism (PSAT1, PSPH, and PHGDH) were differentially expressed. Addition of 10 mM glycine to the culture medium (currently containing 0.1 mM) reduced the abundance of SLC6A9 transcript and increased total cell number, primarily in the trophectoderm lineage (P = 0.003); this was likely by decreasing the percentage of apoptotic nuclei. As serine and glycine can be reversibly metabolized by serine hydroxymethyltransferase 2 (SHMT2), we assessed the abundance of SHMT2 transcript as well as its functional role by inhibiting it with aminomethylphosphonic acid (AMPA), a glycine analog, during in vitro culture. Both AMPA supplementation and elevated glycine decreased the mRNA abundance of SHMT2 and tumor protein p53 (TP53), which is activated in response to cellular stress, compared to controls (P ≤ 0.02). On the other hand, mitochondrial activity of blastocysts, mtDNA copy number, and abundance of mitochondria-related transcripts did not differ between control and 10 mM glycine culture conditions. Despite improvements to these metrics of blastocyst quality, transfer of embryos cultured in 10 mM glycine did not result in pregnancy whereas the transfer of in vitro-produced embryos cultured in control medium yielded live births. Mol. Reprod. Dev. 83: 246-258, 2016. © 2016 The Authors.