Novel off-Targeting Events Identified after Genome Wide Analysis of CRISPR-Cas Edited Pigs.

CRISPR-Cas technology has transformed our ability to introduce targeted modifications, allowing unconventional animal models such as pigs to model human diseases and improve its value for food production. The main concern with using the technology is the possibility of introducing unwanted modifications in the genome. In this study, we illustrate a pipeline to comprehensively identify off-targeting events on a global scale in the genome of three different gene-edited pig models. Whole genome sequencing paired with an off-targeting prediction software tool filtered off-targeting events amongst natural variations present in gene-edited pigs. This pipeline confirmed two known off-targeting events in IGH knockout pigs, AR and RBFOX1, and identified other presumably off-targeted loci. Independent validation of the off-targeting events using other gene-edited DNA confirmed two novel off-targeting events in RAG2/IL2RG knockout pig models. This unique strategy offers a novel tool to detect off-targeting events in genetically heterogeneous species after genome editing.

Porcine-specific expression of the three functional CYP19 paralogs in early conceptus, placenta, and gonads.

In brief: Aromatase catalyzes the synthesis of estrogens and has been shown to have an important role during the establishment of pregnancy in the pig. This study confirmed the differential expression of the three aromatase isoforms. Abstract: Although three porcine aromatase isoforms have been identified, their gene expression profiles in reproduction are still poorly understood. Here, we identified by Sanger sequencing unique nucleotide signatures for the three paralogous copies of Cyp19 and analyzed by RT-PCR the occurrence of the Cyp19 and Cyp17a1 transcripts at different tissues and stages of conceptus and fetal-placental development. Cyp19a1 and Cyp19a3 expressions were detected in conceptuses and gonads, respectively. Cyp19a2 transcripts were identified on both the conceptuses and the placenta samples. Transcripts for Cyp17a1 were detected predominantly in conceptus and gonads. In the endometrium of day 21 pregnant females, as well as days 12 and 17 pseudopregnant females, we did not detect the expression of Cyp19a1, Cyp19a2, or Cyp19a3. In our study, we have demonstrated distinct transcriptional regulation for the three functional Cyp19 paralogs and a potential role for Cyp17a1 in controlling the secretion of estrogen from the conceptus and the placenta.

Inactivation of growth differentiation factor 9 blocks folliculogenesis in pigs†.

Growth differentiation factor 9 (GDF9) is a secreted protein belonging to the transforming growth factor beta superfamily and has been well characterized for its role during folliculogenesis in the ovary. Although previous studies in mice and sheep have shown that mutations in GDF9 disrupt follicular progression, the exact role of GDF9 in pigs has yet to be elucidated. The objective of this study was to understand the role of GDF9 in ovarian function by rapidly generating GDF9 knockout (GDF9-/-) pigs by using the CRISPR/Cas9 system. Three single-guide RNAs designed to disrupt porcine GDF9 were injected with Cas9 mRNA into zygotes, and blastocyst-stage embryos were transferred into surrogates. One pregnancy was sacrificed on day 100 of gestation to investigate the role of GDF9 during oogenesis. Four female fetuses were recovered with one predicted to be GDF9-/- and the others with in-frame mutations. All four had fully formed oocytes within primordial follicles, confirming that knockout of GDF9 does not disrupt oogenesis. Four GDF9 mutant gilts were generated and were grown past puberty. One gilt was predicted to completely lack functional GDF9 (GDF9-/-), and the gilt never demonstrated standing estrus and had a severely underdeveloped reproductive tract with large ovarian cysts. Further examination revealed that the follicles from the GDF9-/- gilt did not progress past preantral stages, and the uterine vasculature was less extensive than the control pigs. By using the CRISPR/Cas9 system, we demonstrated that GDF9 is a critical growth factor for proper ovarian development and function in pigs.

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.

Challenges and Considerations during In Vitro Production of Porcine Embryos.

Genetically modified pigs have become valuable tools for generating advances in animal agriculture and human medicine. Importantly, in vitro production and manipulation of embryos is an essential step in the process of creating porcine models. As the in vitro environment is still suboptimal, it is imperative to examine the porcine embryo culture system from several angles to identify methods for improvement. Understanding metabolic characteristics of porcine embryos and considering comparisons with other mammalian species is useful for optimizing culture media formulations. Furthermore, stressors arising from the environment and maternal or paternal factors must be taken into consideration to produce healthy embryos in vitro. In this review, we progress stepwise through in vitro oocyte maturation, fertilization, and embryo culture in pigs to assess the status of current culture systems and address points where improvements can be made.

Conceptus interferon gamma is essential for establishment of pregnancy in the pig†.

Establishment and maintenance of pregnancy in the pig is a complex process that relies on conceptus regulation of the maternal proinflammatory response to endometrial attachment. Following elongation, pig conceptuses secrete interferon gamma (IFNG) during attachment to the endometrial luminal epithelium. The objective here was to determine if conceptus production of IFNG is important for early development and establishment of pregnancy. CRISPR/Cas9 gene editing and somatic cell nuclear transfer technologies were used to create an IFNG loss-of-function study in pigs. Wild-type (IFNG+/+) and null (IFNG-/-) fibroblast cells were used to create embryos through somatic cell nuclear transfer. IFNG expression was not detected in IFNG-/- conceptuses on either day 15 or day 17 of pregnancy. Ablation of conceptus IFNG production resulted in the reduction of stromal CD3+ and mast cells, which localized to the site of conceptus attachment on day 15. The uteri of recipients with IFNG-/- conceptuses were inflamed, hyperemic and there was an abundance of erythrocytes in the uterine lumen associated with the degenerating conceptuses. The endometrial stromal extracellular matrix was altered in the IFNG-/- embryo pregnancies and there was an increased endometrial mRNA levels for collagen XVII (COL17A1), matrilin 1 (MATN1), secreted phosphoprotein 1 (SPP1), and cysteine-rich secretory protein 3 (CRISP3), which are involved with repair and remodeling of the extracellular matrix. These results indicate conceptus IFNG production is essential in modulating the endometrial proinflammatory response for conceptus attachment and survival in pigs.

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.

Glutaminolysis is involved in the activation of mTORC1 in in vitro-produced porcine embryos.

Glutamine supplementation to porcine embryo culture medium improves development, increases leucine consumption, and enhances mitochondrial activity. In cancer cells, glutamine has been implicated in the activation of mechanistic target of rapamycin complex 1 (mTORC1) to support rapid proliferation. The objective of this study was to determine if glutamine metabolism, known as glutaminolysis, was involved in mTORC1 activation in porcine embryos. Culture with 3.75 mM GlutaMAX improved development to the blastocyst stage compared to culture with 1 mM GlutaMAX, and culture with 0 mM GlutaMAX decreased development compared to all groups with GlutaMAX. Ratios of phosphorylated to total MTOR were increased when embryos were cultured with 3.75 or 10 mM GlutaMAX, which was enhanced by the absence of leucine, but ratios for RPS6K were unchanged. As another indicator of mTORC1 activation, colocalization of MTOR and a lysosomal marker was increased in embryos cultured with 3.75 or 10 mM GlutaMAX in the absence of leucine. Culturing embryos with glutaminase inhibitors decreased development and the ratio of phosphorylated to total MTOR, indicating reduced activation of the complex. Therefore, glutaminolysis is involved in the activation of mTORC1 in porcine embryos, but further studies are needed to characterize downstream effects on development.

Chemical simulation of hypoxia in donor cells improves development of somatic cell nuclear transfer-derived embryos and increases abundance of transcripts related to glycolysis.

To improve efficiency of somatic cell nuclear transfer (SCNT), it is necessary to modify differentiated donor cells to become more amendable for reprogramming by the oocyte cytoplasm. A key feature that distinguishes somatic/differentiated cells from embryonic/undifferentiated cells is cellular metabolism, with somatic cells using oxidative phosphorylation (OXPHOS) while embryonic cells utilize glycolysis. Inducing metabolic reprogramming in donor cells could improve SCNT efficiency by priming cells to become more embryonic in nature before SCNT hypoxia inducible factor 1-α (HIF1-α), a transcription factor that allows for cell survival in low oxygen, promotes a metabolic switch from OXPHOS to glycolysis. We hypothesized that chemically stabilizing HIF1-α in donor cells by use of the hypoxia mimetic, cobalt chloride (CoCl2 ), would promote this metabolic switch in donor cells and subsequently improve the development of SCNT embryos. Donor cell treatment with 100 µM CoCl2 for 24 hr preceding SCNT upregulated messenfer RNA abundance of glycolytic enzymes, improved SCNT development to the blastocyst stage and quality, and affected gene expression in the blastocysts. After transferring blastocysts created from CoCl2 -treated donor cells to surrogates, healthy cloned piglets were produced. Therefore, shifting metabolism toward glycolysis in donor cells by CoCl2 treatment is a simple, economical way of improving the in vitro efficiency of SCNT and is capable of producing live animals.

Removal of hypotaurine from porcine embryo culture medium does not impair development of in vitro-fertilized or somatic cell nuclear transfer-derived embryos at low oxygen tension.

Hypotaurine (HT) is a routine component of porcine embryo culture medium, functioning as an antioxidant, but its requirement may be diminished as most embryo culture systems now use 5% O2 instead of atmospheric (20%) O2 . Our objective was to determine the effects of removing HT from the culture medium on porcine preimplantation embryo development. Embryos cultured in 20% O2 without HT had decreased blastocyst development compared to culture with HT or in 5% O2 with or without HT. Notably, differences in blastocyst development or total cell number were not detected between embryos cultured in 5% O2 with or without HT. After culture in 5% O2 without HT and embryo transfer, healthy fetuses were retrieved from two pregnancies on Day 42, confirming in vivo developmental competence. Transcript abundance of proapoptotic markers was decreased in embryos cultured without HT regardless of oxygen tension; however, assays for apoptosis did not demonstrate differences between groups. Additionally, no differences were observed in the development or apoptosis of somatic cell nuclear transfer-derived embryos cultured in 5% O2 with or without HT. With decreased utility in 5% O2 , removing HT from porcine embryo culture medium would also have economic advantages because it is undoubtedly the most expensive component.

Ablation of conceptus PTGS2 expression does not alter early conceptus development and establishment of pregnancy in the pig†.

Pig conceptuses secrete estrogens (E2), interleukin 1 beta 2 (IL1B2), and prostaglandins (PGs) during the period of rapid trophoblast elongation and establishment of pregnancy. Previous studies established that IL1B2 is essential for rapid conceptus elongation, whereas E2 is not essential for conceptus elongation or early maintenance of the corpora lutea. The objective of the present study was to determine if conceptus expression of prostaglandin-endoperoxide synthase 2 (PTGS2) and release of PG are important for early development and establishment of pregnancy. To understand the role of PTGS2 in conceptus elongation and pregnancy establishment, a loss-of-function study was conducted by editing PTGS2 using CRISPR/Cas9 technology. Wild-type (PTGS2+/+) and null (PTGS2-/-) fibroblast cells were used to create embryos through somatic cell nuclear transfer. Immunolocalization of PTGS2 and PG production was absent in cultured PTGS2-/- blastocysts on day 7. PTGS2+/+ and PTGS2-/- blastocysts were transferred into surrogate gilts, and the reproductive tracts were collected on either days 14, 17, or 35 of pregnancy. After flushing the uterus on days 14 and 17, filamentous conceptuses were cultured for 3 h to determine PG production. Conceptus release of total PG, prostaglandin F2⍺ (PGF2α), and PGE in culture media was lower with PTGS2-/- conceptuses compared to PTGS2+/+ conceptuses. However, the total PG, PGF2α, and PGE content in the uterine flushings was not different. PTGS2-/- conceptus surrogates allowed to continue pregnancy were maintained beyond 30 days of gestation. These results indicate that pig conceptus PTGS2 is not essential for early development and establishment of pregnancy in the pig.

Applications of omics and nanotechnology to improve pig embryo production in vitro.

An appropriate environment to optimize porcine preimplantation embryo production in vitro is required as genetically modified pigs have become indispensable for biomedical research and agriculture. To provide suitable culture conditions, omics technologies have been applied to elucidate which metabolic substrates and pathways are involved during early developmental processes. Metabolomic profiling and transcriptional analysis comparing in vivo- and in vitro-derived embryos have demonstrated the important role of amino acids during preimplantation development. Transcriptional profiling studies have been helpful in assessing epigenetic reprogramming agents to allow for the correction of gene expression during the cloning process. Along with this, nanotechnology, which is a highly promising field, has allowed for the use of engineered nanoplatforms in reproductive biology. A growing number of studies have explored the use of nanoengineered materials for sorting, labeling, and targeting purposes; which demonstrates their potential to become one of the solutions for precise delivery of molecules into gametes and embryos. Considering the contributions of omics and the recent progress in nanoscience, in this review, we focused on their emerging applications for current in vitro pig embryo production systems to optimize the generation of genetically modified animals.

Glutamine supplementation enhances development of in vitro-produced porcine embryos and increases leucine consumption from the medium.

Improper composition of culture medium contributes to reduced viability of in vitro-produced embryos. Glutamine (Gln) is a crucial amino acid for preimplantation embryos as it supports proliferation and is involved in many different biosynthetic pathways. Previous transcriptional profiling revealed several upregulated genes related to Gln transport and metabolism in in vitro-produced porcine blastocysts compared to in vivo-produced counterparts, indicating a potential deficiency in the culture medium. Therefore, the objective of this study was to determine the effects of Gln supplementation on in vitro-produced porcine embryo development, gene expression, and metabolism. Cleaved embryos were selected and cultured in MU2 medium supplemented with 1 mM Gln (control), 3.75 mM Gln (+Gln), 3.75 mM GlutaMAX (+Max), or 3.75 mM alanine (+Ala) until day 6. Embryos cultured with +Gln or +Max had increased development to the blastocyst stage and total number of nuclei compared to the control (P < 0.05). Moreover, expression of misregulated transcripts involved in glutamine and glutamate transport and metabolism was corrected when embryos were cultured with +Gln or +Max. Metabolomics analysis revealed increased production of glutamine and glutamate into the medium by embryos cultured with +Max and increased consumption of leucine by embryos cultured with +Gln or +Max. As an indicator of cellular health, mitochondrial membrane potential was increased when embryos were cultured with +Max which was coincident with decreased apoptosis in these blastocysts. Lastly, two embryo transfers by using embryos cultured with +Max resulted in viable piglets, confirming that this treatment is consistent with in vivo developmental competence.

Identification of the MUC2 Promoter as a Strong Promoter for Intestinal Gene Expression through Generation of Transgenic Quail Expressing GFP in Gut Epithelial Cells.

Identification of tissue- and stage-specific gene promoters is valuable for delineating the functional roles of specific genes in genetically engineered animals. Here, through the comparison of gene expression in different tissues by analysis of a microarray database, the intestinal specificity of mucin 2 (MUC2) expression was identified in mice and humans, and further confirmed in chickens by RT-PCR (reverse transcription-PCR) analysis. An analysis of cis-acting elements in avian MUC2 gene promoters revealed conservation of binding sites, within a 2.9 kb proximal promoter region, for transcription factors such as caudal type homeobox 2 (CDX2), GATA binding protein 4 (GATA4), hepatocyte nuclear factor 4 α (HNF4A), and transcription factor 4 (TCF4) that are important for maintaining intestinal homeostasis and functional integrity. By generating transgenic quail, we demonstrated that the 2.9 kb chicken MUC2 promoter could drive green fluorescent protein (GFP) reporter expression exclusively in the small intestine, large intestine, and ceca. Fluorescence image analysis further revealed GFP expression in intestine epithelial cells. The GFP expression was barely detectable in the embryonic intestine, but increased during post-hatch development. The spatiotemporal expression pattern of the reporter gene confirmed that the 2.9 kb MUC2 promoter could retain the regulatory element to drive expression of target genes in intestinal tissues after hatching. This new transgene expression system, using the MUC2 promoter, will provide a new method of overexpressing target genes to study gene function in the avian intestine.