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.

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.

A porcine model of phenylketonuria generated by CRISPR/Cas9 genome editing.

Phenylalanine hydroxylase-deficient (PAH-deficient) phenylketonuria (PKU) results in systemic hyperphenylalaninemia, leading to neurotoxicity with severe developmental disabilities. Dietary phenylalanine (Phe) restriction prevents the most deleterious effects of hyperphenylalaninemia, but adherence to diet is poor in adult and adolescent patients, resulting in characteristic neurobehavioral phenotypes. Thus, an urgent need exists for new treatments. Additionally, rodent models of PKU do not adequately reflect neurocognitive phenotypes, and thus there is a need for improved animal models. To this end, we have developed PAH-null pigs. After selection of optimal CRISPR/Cas9 genome-editing reagents by using an in vitro cell model, zygote injection of 2 sgRNAs and Cas9 mRNA demonstrated deletions in preimplantation embryos, with embryo transfer to a surrogate leading to 2 founder animals. One pig was heterozygous for a PAH exon 6 deletion allele, while the other was compound heterozygous for deletions of exon 6 and of exons 6-7. The affected pig exhibited hyperphenylalaninemia (2000-5000 µM) that was treatable by dietary Phe restriction, consistent with classical PKU, along with juvenile growth retardation, hypopigmentation, ventriculomegaly, and decreased brain gray matter volume. In conclusion, we have established a large-animal preclinical model of PKU to investigate pathophysiology and to assess new therapeutic interventions.

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.

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 .

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.

Single step production of Cas9 mRNA for zygote injection.

Production of Cas9 mRNA in vitro typically requires the addition of a 5´ cap and 3´ polyadenylation. A plasmid was constructed that harbored the T7 promoter followed by the EMCV IRES and a Cas9 coding region. We hypothesized that the use of the metastasis associated lung adenocarcinoma transcript 1 (Malat1) triplex structure downstream of an IRES/Cas9 expression cassette would make polyadenylation of in vitro produced mRNA unnecessary. A sequence from the mMalat1 gene was cloned downstream of the IRES/Cas9 cassette described above. An mRNA concentration curve was constructed with either commercially available Cas9 mRNA or the IRES/ Cas9/triplex, by injection into porcine zygotes. Blastocysts were genotyped to determine if differences existed in the percent of embryos modified. The concentration curve identified differences due to concentration and RNA type injected. Single step production of Cas9 mRNA provides an alternative source of Cas9 for use in zygote injections.

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.

Pharmacologic treatment of donor cells induced to have a Warburg effect-like metabolism does not alter embryonic development in vitro or survival during early gestation when used in somatic cell nuclear transfer in pigs.

Somatic cell nuclear transfer is a valuable technique for the generation of genetically engineered animals, however, the efficiency of cloning in mammalian species is low (1-3%). Differentiated somatic cells commonly used in nuclear transfer utilize the tricarboxylic acid cycle and cellular respiration for energy production. Comparatively the metabolism of somatic cells contrasts that of the cells within the early embryos which predominately use glycolysis. Early embryos (prior to implantation) are evidenced to exhibit characteristics of a Warburg Effect (WE)-like metabolism. We hypothesized that pharmacologically driven fibroblast cells can become more blastomere-like and result in improved in vitro embryonic development after SCNT. The goals were to determine if subsequent in vitro embryo development is impacted by (1) cloning pharmacologically treated donor cells pushed to have a WE-like metabolism or (2) culturing non-treated donor clones with pharmaceuticals used to push a WE-like metabolism. Additionally, we investigated early gestational survival of the donor-treated clone embryos. Here we demonstrate that in vitro development of clones is not hindered by pharmacologically treating either the donor cells or the embryos themselves with CPI, PS48, or the combination of these drugs. Furthermore, these experiments demonstrate that early embryos (or at least in vitro produced embryos) have a low proportion of mitochondria which have high membrane potential and treatment with these pharmaceuticals does not further alter the mitochondrial function in early embryos. Lastly, we show that survival in early gestation was not different between clones from pharmacologically induced WE-like donor cells and controls.

Inactivation of porcine interleukin-1ß results in failure of rapid conceptus elongation.

Conceptus expansion throughout the uterus of mammalian species with a noninvasive epitheliochorial type of placentation is critical establishing an adequate uterine surface area for nutrient support during gestation. Pig conceptuses undergo a unique rapid morphological transformation to elongate into filamentous threads within 1 h, which provides the uterine surface to support development and maintain functional corpora lutea through the production of estrogen. Conceptus production of a unique interleukin 1ß, IL1B2, temporally increases during the period of trophoblast remodeling during elongation. CRISPR/Cas9 gene editing was used to knock out pig conceptus IL1B2 expression and the secretion of IL1B2 during the time of conceptus elongation. Trophoblast elongation occurred on day 14 in wild-type (IL1B2+/+) conceptuses but did not occur in ILB2-null (IL1B2-/-) conceptuses. Although the morphological transition of IL1B2-/- conceptuses was inhibited, expression of a number of conceptus developmental genes was not altered. However, conceptus aromatase expression and estrogen secretion were decreased, indicating that IL1B2 may be involved in the spatiotemporal increase in conceptus estrogen synthesis needed for the establishment of pregnancy in the pig and may serve to regulate the proinflammatory response of endometrium to IL1B2 during conceptus elongation and attachment to the uterine surface.

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.

Inclusion of homologous DNA in nuclease-mediated gene targeting facilitates a higher incidence of bi-allelically modified cells.

BACKGROUND: Recent advancements in gene editing techniques have increased in number and utility. These techniques are an attractive alternative to conventional gene targeting methods via homologous recombination due to the ease of use and the high efficiency of gene editing. We have previously produced cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) knockout (KO) pigs in a Minnesota miniature pig genetic background. These pigs were generated using zinc-finger nucleases (ZFNs) in combination with donor DNA containing a total homology length of 1600 bp (800-bp homology on each arm). Our next aim was to introduce the targeted disruption of alpha-1,3-galactosyltransferase (GGTA1) in the CMAH KO genetic background and evaluate the effect of donor DNA homology length on meganuclease-mediated gene targeting. METHODS: Zinc-finger nucleases from a previous CMAH KO experiment were used as a proof of concept to identify a correlation between the length of donor DNA homology and targeting efficiency. Based on those results, experiments were designed to use transcription activator-like effector nucleases (TALENs) to generate bi-allelically modified GGTA1 cells using donor DNAs carrying various lengths of homology. Donor DNA was designed to symmetrically flank the predicted cleavage sites in CMAH and GGTA1 for both ZFN and TALEN cleavage sites, respectively. For both genes, the length of total homology ranged from 60 to 1799 bp. Sialyltransferase gene expression profiles were evaluated in CMAH and GGTA1 double KO pig cells and were compared to wild-type and CMAH KO cells. RESULTS: Introduction of donor DNA with ZFNs demonstrated that small amounts of homology (60 bp) could facilitate homology-directed repair during ZFN-mediated targeting of CMAH; however, donor DNA with longer amounts of homology resulted in a higher frequency of homology-directed repair. For the GGTA1 KO experiments that used TALENs and donor DNA, donor DNA alone did not result in detectable bi-allelic conversion of GGTA1. As the length of donor DNA increased, the bi-allelic disruption of GGTA1 increased from 0.5% (TALENs alone, no donor DNA present) to a maximum of 3% (TALENs and donor DNA with total homology of 1799 bp). Inclusion of homologous donor DNA in TALEN-mediated gene targeting facilitated a higher incidence of bi-allelically modified cells. Using the generated cells, we were able to demonstrate the lack of GGTA1 expression and the decrease in gene expression sialyltransferase-related genes. CONCLUSIONS: The approach of using donor DNA in conjunction with a meganuclease can be used to increase the efficiency of gene targeting. The gene editing methods can be applied to other genes as well as other mammalian systems. Additionally, gene expression analysis further confirms that the CMAH/GGTA1 double KO pigs can be a valuable source for the study of pig-to-human xenotransplantation.

Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos.

Targeted modification of the pig genome can be challenging. Recent applications of the CRISPR/Cas9 system hold promise for improving the efficacy of genome editing. When a designed CRISPR/Cas9 system targeting CD163 or CD1D was introduced into somatic cells, it was highly efficient in inducing mutations. When these mutated cells were used with somatic cell nuclear transfer, offspring with these modifications were created. When the CRISPR/Cas9 system was delivered into in vitro produced presumptive porcine zygotes, the system was effective in creating mutations in eGFP, CD163, and CD1D (100% targeting efficiency in blastocyst stage embryos); however, it also presented some embryo toxicity. We could also induce deletions in CD163 or CD1D by introducing two types of CRISPRs with Cas9. The system could also disrupt two genes, CD163 and eGFP, simultaneously when two CRISPRs targeting two genes with Cas9 were delivered into zygotes. Direct injection of CRISPR/Cas9 targeting CD163 or CD1D into zygotes resulted in piglets that have mutations on both alleles with only one CD1D pig having a mosaic genotype. We show here that the CRISPR/Cas9 system can be used by two methods. The system can be used to modify somatic cells followed by somatic cell nuclear transfer. System components can also be used in in vitro produced zygotes to generate pigs with specific genetic modifications.