Reconstitution of UCP1 using CRISPR/Cas9 in the white adipose tissue of pigs decreases fat deposition and improves thermogenic capacity.

Uncoupling protein 1 (UCP1) is localized on the inner mitochondrial membrane and generates heat by uncoupling ATP synthesis from proton transit across the inner membrane. UCP1 is a key element of nonshivering thermogenesis and is most likely important in the regulation of body adiposity. Pigs (Artiodactyl family Suidae) lack a functional UCP1 gene, resulting in poor thermoregulation and susceptibility to cold, which is an economic and pig welfare issue owing to neonatal mortality. Pigs also have a tendency toward fat accumulation, which may be linked to their lack of UCP1, and thus influences the efficiency of pig production. Here, we report application of a CRISPR/Cas9-mediated, homologous recombination (HR)-independent approach to efficiently insert mouse adiponectin-UCP1 into the porcine endogenous UCP1 locus. The resultant UCP1 knock-in (KI) pigs showed an improved ability to maintain body temperature during acute cold exposure, but they did not have alterations in physical activity levels or total daily energy expenditure (DEE). Furthermore, ectopic UCP1 expression in white adipose tissue (WAT) dramatically decreased fat deposition by 4.89% (P < 0.01), consequently increasing carcass lean percentage (CLP; P < 0.05). Mechanism studies indicated that the loss of fat upon UCP1 activation in WAT was linked to elevated lipolysis. UCP1 KI pigs are a potentially valuable resource for agricultural production through their combination of cold adaptation, which improves pig welfare and reduces economic losses, with reduced fat deposition and increased lean meat production.

Thyroid hormone regulates hematopoiesis via the TR-KLF9 axis.

Congenital hypothyroidism (CH) is one of the most prevalent endocrine diseases, for which the underlying mechanisms remain unknown; it is often accompanied by anemia and immunodeficiency in patients. Here, we created a severe CH model together with anemia and T lymphopenia to mimic the clinical features of hypothyroid patients by ethylnitrosourea (ENU) mutagenesis in Bama miniature pigs. A novel recessive c.1226A>G transition of the dual oxidase 2 (DUOX2) gene was identified as the causative mutation. This mutation hindered the production of hydrogen peroxide (H2O2) and thus contributed to thyroid hormone (TH) synthesis failure. Transcriptome sequencing analysis of the thymuses showed that Krüppel-like factor 9 (KLF9) was predominantly downregulated in hypothyroid mutants. KLF9 was verified to be directly regulated by TH in a TH receptor (TR)-dependent manner both in vivo and in vitro. Furthermore, knockdown of klf9 in zebrafish embryos impaired hematopoietic development including erythroid maturation and T lymphopoiesis. Our findings suggest that the TR-KLF9 axis is responsible for the hematopoietic dysfunction and might be exploited for the development of novel therapeutic interventions for thyroid diseases.

Creation of miniature pig model of human Waardenburg syndrome type 2A by ENU mutagenesis.

Human Waardenburg syndrome 2A (WS2A) is a dominant hearing loss (HL) syndrome caused by mutations in the microphthalmia-associated transcription factor (MITF) gene. In mouse models with MITF mutations, WS2A is transmitted in a recessive pattern, which limits the study of hearing loss (HL) pathology. In the current study, we performed ENU (ethylnitrosourea) mutagenesis that resulted in substituting a conserved lysine with a serine (p. L247S) in the DNA-binding domain of the MITF gene to generate a novel miniature pig model of WS2A. The heterozygous mutant pig (MITF +/L247S) exhibits a dominant form of profound HL and hypopigmentation in skin, hair, and iris, accompanied by degeneration of stria vascularis (SV), fused hair cells, and the absence of endocochlear potential, which indicate the pathology of human WS2A. Besides hypopigmentation and bilateral HL, the homozygous mutant pig (MITF L247S/L247S) and CRISPR/Cas9-mediated MITF bi-allelic knockout pigs both exhibited anophthalmia. Three WS2 patients carrying MITF mutations adjacent to the corresponding region were also identified. The pig models resemble the clinical symptom and molecular pathology of human WS2A patients perfectly, which will provide new clues for better understanding the etiology and development of novel treatment strategies for human HL.

BIX-01294 increases pig cloning efficiency by improving epigenetic reprogramming of somatic cell nuclei.

Accumulating evidence suggests that faulty epigenetic reprogramming leads to the abnormal development of cloned embryos and results in the low success rates observed in all mammals produced through somatic cell nuclear transfer (SCNT). The aberrant methylation status of H3K9me and H3K9me2 has been reported in cloned mouse embryos. To explore the role of H3K9me2 and H3K9me in the porcine somatic cell nuclear reprogramming, BIX-01294, known as a specific inhibitor of G9A (histone-lysine methyltransferase of H3K9), was used to treat the nuclear-transferred (NT) oocytes for 14-16 h after activation. The results showed that the developmental competence of porcine SCNT embryos was significantly enhanced both in vitro (blastocyst rate 16.4% vs 23.2%, P<0.05) and in vivo (cloning rate 1.59% vs 2.96%) after 50 nm BIX-01294 treatment. BIX-01294 treatment significantly decreased the levels of H3K9me2 and H3K9me at the 2- and 4-cell stages, which are associated with embryo genetic activation, and increased the transcriptional expression of the pluripotency genes SOX2, NANOG and OCT4 in cloned blastocysts. Furthermore, the histone acetylation levels of H3K9, H4K8 and H4K12 in cloned embryos were decreased after BIX-01294 treatment. However, co-treatment of activated NT oocytes with BIX-01294 and Scriptaid rescued donor nuclear chromatin from decreased histone acetylation of H4K8 that resulted from exposure to BIX-01294 only and consequently improved the preimplantation development of SCNT embryos (blastocyst formation rates of 23.7% vs 21.5%). These results indicated that treatment with BIX-01294 enhanced the developmental competence of porcine SCNT embryos through improvements in epigenetic reprogramming and gene expression.

LepRb+ cell-specific deletion of Slug mitigates obesity and nonalcoholic fatty liver disease in mice.

Leptin exerts its biological actions by activating the long-form leptin receptor (LepRb). LepRb signaling impairment and leptin resistance are believed to cause obesity. The transcription factor Slug - also known as Snai2 - recruits epigenetic modifiers and regulates gene expression by an epigenetic mechanism; however, its epigenetic action has not been explored in leptin resistance. Here, we uncover a proobesity function of neuronal Slug. Hypothalamic Slug was upregulated in obese mice. LepRb+ cell-specific Slug-knockout (SlugΔLepRb) mice were resistant to diet-induced obesity, type 2 diabetes, and liver steatosis and experienced decreased food intake and increased fat thermogenesis. Leptin stimulated hypothalamic Stat3 phosphorylation and weight loss to a markedly higher level in SlugΔLepRb than in Slugfl/fl mice, even before their body weight divergence. Conversely, hypothalamic LepRb+ neuron-specific overexpression of Slug, mediated by AAV-hSyn-DIO-Slug transduction, induced leptin resistance, obesity, and metabolic disorders in mice on a chow diet. At the genomic level, Slug bound to and repressed the LepRb promoter, thereby inhibiting LepRb transcription. Consistently, Slug deficiency decreased methylation of LepRb promoter H3K27, a repressive epigenetic mark, and increased LepRb mRNA levels in the hypothalamus. Collectively, these results unravel what we believe to be a previously unrecognized hypothalamic neuronal Slug/epigenetic reprogramming/leptin resistance axis that promotes energy imbalance, obesity, and metabolic disease.

Fecal Microbiota Was Reshaped in UCP1 Knock-In Pigs via the Adipose-Liver-Gut Axis and Contributed to Less Fat Deposition.

The relationship between the host gut microbiota and obesity has been well documented in humans and mice; however, few studies reported the association between the gut microbiota and fat deposition in pigs. In a previous study, we generated uncoupling protein 1 (UCP1) knock-in pigs (UCP1 pigs), which exhibited a lower fat deposition phenotype. Whether the gut microbiota was reshaped in these pigs and whether the reshaped gut microbiota contributes to the lower fat content remain unknown. Here, we revealed that the fecal microbiota composition and metabolites were significantly altered under both chow diet (CD) and high-fat/high-cholesterol (HFHC) diet conditions in UCP1 pigs compared to those in wild-type (WT) pigs. The abundance of Oscillospira and Coprococcus and the level of metabolite hyodeoxycholic acid (HDCA) from feces were observed to be significantly increased in UCP1 pigs. An association analysis revealed that Oscillospira and Coprococcus were significantly negatively related to backfat thickness. In addition, after fecal microbiota transplantation (FMT), the mice that were orally gavaged with feces from UCP1 pigs exhibited less fat deposition under both CD and high-fat diet (HFD) conditions, suggesting that the fecal microbes of UCP1 pigs participate in regulating host lipid metabolism. Consistently, HDCA-treated mice also exhibited reduced fat content. Mechanistically, we found that UCP1 expression in white adipose tissue alters the gut microbiota via the adipose-liver-gut axis in pigs. Our study provides new data concerning the cross talk between host genetic variations and the gut microbiota and paves the way for the potential application of microbes or their metabolites in the regulation of fat deposition in pigs. IMPORTANCE This article investigated the effect of the ectopic expression of UCP1 on the regulation of fecal microbiota composition and metabolites and which alters the fat deposition phenotype. Bacteria, including Oscillospira and Coprococcus, and the metabolite HDCA were found to be significantly increased in feces of UCP1 pigs and had a negative relationship with backfat thickness. Mice with fecal microbiota transplantation phenocopied the UCP1 pigs under both CD and HFD conditions, suggesting that the fecal microbes of UCP1 pigs participate in regulating host lipid metabolism. Our study provides new data regarding the cross talk between host genetic variations and the gut microbiota and paves the way for the potential application of microbes or their metabolic production in the regulation of fat deposition in pigs.

One-step base editing in multiple genes by direct embryo injection for pig trait improvement.

The precise and simultaneous acquisition of multiple beneficial alleles in the genome is in great demand for the development of elite pig breeders. Cytidine base editors (CBEs) that convert C:G to T:A have emerged as powerful tools for single-nucleotide replacement. Whether CBEs can effectively mediate C-to-T substitution at multiple sites/loci for trait improvement by direct zygote injection has not been verified in large animals. Here, we determined the editing efficiency of four CBE variants in porcine embryonic fibroblast cells and embryos. The findings showed that hA3A-BE3-Y130F and hA3A-eBE-Y130F consistently resulted in increased base-editing efficiency and low toxic effects in embryonic development. Further, we verified that using a one-step approach, direct zygote microinjection of the CBE system can generate pigs harboring multiple point mutations. Our process resulted in a stop codon in CD163 and myostatin (MSTN) and introduced a beneficial allele in insulin-like growth factor-2 (IGF2). The pigs showed disrupted expression of CD163 and MSTN and increased expression of IGF2, which significantly improved growth performance and infectious disease resistance. Our approach allows immediate introduction of multiple mutations in transgene-free animals to comprehensively improve economic traits through direct embryo microinjection, providing a potential new route to produce elite pig breeders.

Mitochondrial uncoupling protein 1 antagonizes atherosclerosis by blocking NLRP3 inflammasome-dependent interleukin-1ß production.

Mitochondrial uncoupling protein 1 (UCP1) is the hallmark of brown adipocytes responsible for cold- and diet-induced thermogenesis. Here, we report a previously unidentified role of UCP1 in maintaining vascular health through its anti-inflammatory actions possibly in perivascular adipose tissue. UCP1 deficiency exacerbates dietary obesity-induced endothelial dysfunction, vascular inflammation, and atherogenesis in mice, which was not rectified by reconstitution of UCP1 in interscapular brown adipose tissue. Mechanistically, lack of UCP1 augments mitochondrial membrane potential and mitochondrial superoxide, leading to hyperactivation of the NLRP3-inflammasome and caspase-1­mediated maturation of interleukin-1ß (IL-1ß). UCP1 deficiency­evoked deterioration of vascular dysfunction and atherogenesis is reversed by IL-1ß neutralization or a chemical mitochondrial uncoupler. Furthermore, UCP1 knockin pigs (which lack endogenous UCP1) are refractory to vascular inflammation and coronary atherosclerosis. Thus, UCP1 acts as a gatekeeper to prevent NLRP3 inflammasome activation and IL-1ß production in the vasculature, thereby conferring a protective effect against cardiovascular diseases.

Cytosine Base Editor (hA3A-BE3-NG)-Mediated Multiple Gene Editing for Pyramid Breeding in Pigs.

Pig is an important agricultural economic animal, providing large amount of meat products. With the development of functional genomics and bioinformatics, lots of genes and functional single nucleotide polymorphisms (SNPs) related to disease resistance and (or) economic traits in pigs have been identified, which provides the targets for genetic improvement by genome editing. Base editors (BEs), combining Cas9 nickase and cytidine or adenine deaminase, achieve all four possible transition mutations (C-to-T, A-to-G, T-to-C, and G-to-A) efficiently and accurately without double strand breaks (DSBs) under the protospacer adjacent motif (PAM) sequence of NGG. However, the NGG PAM in canonical CRISPR-Cas9 can only cover approximately 8.27% in the whole genome which limits its broad application. In the current study, hA3A-BE3-NG system was constructed with the fusion of SpCas9-NG variant and hA3A-BE3 to create C-to-T conversion at NGN PAM sites efficiently. The editing efficiency and scope of hA3A-BE3-NG were confirmed in HEK293T cells and porcine fetal fibroblast (PFF) cells. Results showed that the efficiency of hA3A-BE3-NG was much higher than that of hA3A-BE3 on NGH (H = A, C, or T) PAM sites (21.27 vs. 2.81% at average). Further, nonsense and missense mutations were introduced efficiently and precisely via hA3A-BE3-NG in multiple pig economic trait-related genes (CD163, APN, MSTN, and MC4R) in PFF cells by one transfection. The current work indicates the potential applications of hA3A-BE3-NG for pyramid breeding studies in livestock.

Adipose lipidomics and RNA-Seq analysis revealed the enhanced mitochondrial function in UCP1 knock-in pigs.

Uncoupling protein 1 (UCP1) plays a key role in nonshivering thermogenesis and is involved in the pathogenesis of obesity. In a previous study, we generated adipocyte-specific UCP1 knock-in (UCP1-KI) pigs, which exhibited improved thermoregulatory ability and decreased fat deposition. To investigate whether UCP1 knock-in alters the lipid composition of adipose tissues, lipidomics of inguinal subcutaneous white adipose tissue (iWAT) and backfat from 6-month-old cold-treated UCP1-KI pigs and wild-type (WT) pigs were profiled. In addition, genome-wide RNA-sequencing of iWAT was performed to further study the genetic basis for lipid alterations. The results showed that iWAT and backfat from UCP1-KI pigs exhibited distinct lipidomic profiles, as the mild lipid alteration was observed in backfat of UCP1 knock-in pigs. Inguinal WAT from UCP1-KI pigs contained significantly decreased total triacylglycerol (p < 0.05), together with the downregulation of genes involved in fatty acid metabolism, suggesting the decreased lipogenesis in iWAT of UCP1-KI pigs. Significantly increased levels of total sphingolipids (p<0.05) were also observed in iWAT from UCP1-KI pigs. Notably, two mitochondrial-specific lipid species, cardiolipin CL72:8 (18:2) and CL74:9 (18:2), were found to be dramatically increased in iWAT from UCP1-KI pigs, suggesting enhanced mitochondrial function. This observation was further supported by the significant upregulation of numerous mitochondrial-related genes and significantly increased number of large mitochondria and mitochondrial cristae in iWAT of UCP1-KI pigs. Taken together, these data illustrate the specific role of UCP1 in lipid metabolism of fat tissues in pigs and provide new data for characterization of fat traits in UCP1-KI pigs.

A harlequin ichthyosis pig model with a novel ABCA12 mutation can be rescued by acitretin treatment.

Harlequin ichthyosis (HI) is a severe genetic skin disorder and caused by mutation in the ATP-binding cassette A12 (ABCA12) gene. The retinoid administration has dramatically improved long-term survival of HI, but improvements are still needed. However, the ABCA12 null mice failed to respond to retinoid treatment, which impedes the development of novel cure strategies for HI. Here we generated an ethylnitrosourea mutagenic HI pig model (named Z9), which carries a novel deep intronic mutation IVS49-727 A>G in the ABCA12 gene, resulting in abnormal mRNA splicing and truncated protein production. Z9 pigs exhibit significant clinical symptom as human patients with HI. Most importantly, systemic retinoid treatment significantly prolonged the life span of the mutant pigs via improving epidermal maturation, decreasing epidermal apoptosis, and triggering the expression of ABCA6. Taken together, this pig model perfectly resembles the clinical symptom and molecular pathology of patients with HI and will be useful for understanding mechanistic insight and developing therapeutic strategies.

An exonic splicing enhancer mutation in DUOX2 causes aberrant alternative splicing and severe congenital hypothyroidism in Bama pigs.

Pigs share many similarities with humans in terms of anatomy, physiology and genetics, and have long been recognized as important experimental animals in biomedical research. Using an N-ethyl-N-nitrosourea (ENU) mutagenesis screen, we previously identified a large number of pig mutants, which could be further established as human disease models. However, the identification of causative mutations in large animals with great heterogeneity remains a challenging endeavor. Here, we select one pig mutant, showing congenital nude skin and thyroid deficiency in a recessive inheritance pattern. We were able to efficiently map the causative mutation using family-based genome-wide association studies combined with whole-exome sequencing and a small sample size. A loss-of-function variant (c.1226 A>G) that resulted in a highly conserved amino acid substitution (D409G) was identified in the DUOX2 gene. This mutation, located within an exonic splicing enhancer motif, caused aberrant splicing of DUOX2 transcripts and resulted in lower H2O2 production, which might cause a severe defect in thyroid hormone production. Our findings suggest that exome sequencing is an efficient way to map causative mutations and that DUOX2D409G/D409G mutant pigs could be a potential large animal model for human congenital hypothyroidism.

Rescuing ocular development in an anophthalmic pig by blastocyst complementation.

Porcine-derived xenogeneic sources for transplantation are a promising alternative strategy for providing organs for treatment of end-stage organ failure in human patients because of the shortage of human donor organs. The recently developed blastocyst or pluripotent stem cell (PSC) complementation strategy opens a new route for regenerating allogenic organs in miniature pigs. Since the eye is a complicated organ with highly specialized constituent tissues derived from different primordial cell lineages, the development of an intact eye from allogenic cells is a challenging task. Here, combining somatic cell nuclear transfer technology (SCNT) and an anophthalmic pig model (MITFL247S/L247S), allogenic retinal pigmented epithelium cells (RPEs) were retrieved from an E60 chimeric fetus using blastocyst complementation. Furthermore, all structures were successfully regenerated in the intact eye from the injected donor blastomeres. These results clearly demonstrate that not only differentiated functional somatic cells but also a disabled organ with highly specialized constituent tissues can be generated from exogenous blastomeres when delivered to pig embryos with an empty organ niche. This system may also provide novel insights into ocular organogenesis.

Cold adaptation in pigs depends on UCP3 in beige adipocytes.

Pigs lack functional uncoupling protein 1 (UCP1) making them susceptible to cold. Nevertheless, several pig breeds are known to be cold resistant. The molecular mechanism(s) enabling such adaptation are currently unknown. Here, we show that this resistance is not dependent on shivering, but rather depends on UCP3 and white adipose tissue (WAT) browning. In two cold-resistant breeds (Tibetan and Min), but not a cold-sensitive breed (Bama), WAT browning was induced after cold exposure. Beige adipocytes from Tibetan pigs exhibited greater oxidative capacity than those from Bama pigs. Notably, UCP3 expression was significantly increased only in cold-resistant breeds, and knockdown of UCP3 expression in Tibetan adipocytes phenocopied Bama adipocytes in culture. Moreover, the eight dominant pig breeds found across China can be classified into cold-sensitive and cold-resistant breeds based on the UCP3 cDNA sequence. This study indicates that UCP3 has contributed to the evolution of cold resistance in the pig and overturns the orthodoxy that UCP1 is the only thermogenic uncoupling protein.

Pilot study of large-scale production of mutant pigs by ENU mutagenesis.

N-ethyl-N-nitrosourea (ENU) mutagenesis is a powerful tool to generate mutants on a large scale efficiently, and to discover genes with novel functions at the whole-genome level in Caenorhabditis elegans, flies, zebrafish and mice, but it has never been tried in large model animals. We describe a successful systematic three-generation ENU mutagenesis screening in pigs with the establishment of the Chinese Swine Mutagenesis Consortium. A total of 6,770 G1 and 6,800 G3 pigs were screened, 36 dominant and 91 recessive novel pig families with various phenotypes were established. The causative mutations in 10 mutant families were further mapped. As examples, the mutation of SOX10 (R109W) in pig causes inner ear malfunctions and mimics human Mondini dysplasia, and upregulated expression of FBXO32 is associated with congenital splay legs. This study demonstrates the feasibility of artificial random mutagenesis in pigs and opens an avenue for generating a reservoir of mutants for agricultural production and biomedical research.

One-step generation of triple gene-targeted pigs using CRISPR/Cas9 system.

Pig shows multiple superior characteristics in anatomy, physiology, and genome that have made this species to be more suitable models for human diseases, especially for neurodegenerative diseases, because they have similar cerebral convolutions compared with human neocortex. Recently, CRISPR/Cas9 system shows enormous potential for engineering the pig genome. In this study, we expect to generate human Parkinson's disease pig model using CRISPR/Cas9 system by simultaneously targeting three distinct genomic loci, parkin/DJ-1/PINK1, in Bama miniature pigs. By co-injection of Cas9 mRNA and multiplexing single guide RNAs (sgRNAs) targeting parkin, DJ-1, and PINK1 genes, respectively, into in vivo derived pronuclear embryos, we simultaneously targeted three distinct genomic loci. The gene modified piglets remain healthy and display normal behavior at the age of 10 months. In addition, despite the high number of sgRNAs were employed in the present study, our trio-based whole-genome sequencing analysis suggested that the incidence of off-target events is low. Our results demonstrate that the simplicity, efficiency, and power of the CRISPR/Cas9 system to allow for the modification of multiple genes in pigs and yield results of high medical value.

Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs.

Genetic engineering in livestock was greatly enhanced by the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), which can be programmed with a single-guide RNA (sgRNA) to generate site-specific DNA breaks. However, the uncertainties caused by wide variations in sgRNA activity impede the utility of this system in generating genetically modified pigs. Here, we described a single blastocyst genotyping system to provide a simple and rapid solution to evaluate and compare the sgRNA efficiency at inducing indel mutations for a given gene locus. Assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days from the design of the sgRNA. The most effective sgRNA selected by this system was successfully used to induce site-specific insertion through homology-directed repair at a frequency exceeding 13%. Additionally, the highly efficient gene deletion via the selected sgRNA was confirmed in pig fibroblast cells, which could serve as donor cells for somatic cell nuclear transfer. We further showed that direct cytoplasmic injection of Cas9 mRNA and the favorable sgRNA into zygotes could generate biallelic knockout piglets with an efficiency of up to 100%. Thus, our method considerably reduces the uncertainties and expands the practical possibilities of CRISPR/Cas9-mediated genome engineering in pigs.