CREBRF p.Arg457Gln Promotes Obesity and Improves Insulin Sensitivity by Promoting Preadipocyte Differentiation and Reducing Oxidative Metabolism in Gene-modied Pig Models

Obesity is one of the most important risk factors for type 2 diabetes (T2DM). The CREBRF missense allele of rs373863828 (p.Arg457Gln) is associated with increased body mass index (BMI), yet reduced risk of T2DM in people with Pacic ancestry. To investigate the functional consequences of the CREBRF variant, we introduced the corresponding human mutation p.Arg457Gln into porcine genome by using a CRISPR/Cas9-mediated homologous recombination (HR)-dependent approach. The CREBRF p.Arg457Gln pig models displayed dramatically increased fat deposition, yet improved sensitivity to insulin. Transcriptome and metabolome analyses of subcutaneous white adipose tissues showed that the CREBRF p.Arg457Gln mutation promoted preadipocyte differentiation, which indicated that obesity was caused by increased number (hyperplasia) rather than size (hypertrophy) of adipocytes. In addition, the oxidative capacity decreased in the adipose tissue of pigs with CREBRF p.Arg457Gln variant. The preoxidative metabolite content (4-HNE and MDA) signicantly decreased, while activity of antioxidant enzymes (GPX, SOD, and CAT) increased, thereby repressing oxidative metabolism of adipose tissue and reducing level of reactive oxygen species (ROS). The low reactive oxygen species could prevent insulin resistance and reduce risk of obesity-induced type 2 diabetes. This study provides further mechanistic insights into favourable adiposity resulting from CREBRF p.Arg457Gln. old pancreas, intestine, uterus, ovary, musule, adipose .(n=3) f Western blot analysis show no difference of CREBRF expression in WT and CREBRF p.Arg457Gln mutant pigs (one year old adult pigs, n =2). g H&E staining of adipocytes from subcutaneous fat of WT and mutant pigs(one year old adult pigs, n=3). Data showed the adipocytes from mutant pigs is smaller in size and more abundant in number. At least 100 elds of view were observed. (Scale bar: 100 μm.) h The number and diameter of adipocytes in paran section (g) under the same eld were analyzed statistically. At least 100 elds of view were caculated. i For intravenous glucose tolerance tests(IGTT), pigs were fasted for 16 h, and then challenged with intravenous injection of 50% glucose (1.2 ml/kg body weight). Fasting blood glucose levels are lower at 0 min in p.Arg457Gln variant pigs. Blood glucose level in p.Arg457Gln variant pigs rised rapidly in about 15 minutes, then quickly returned to lower levels than wild type pigs. Pigs are one year old. j For insulin tolerance tests(ITT), pigs were fasted for 12 h and then given an intraperitoneal injection of insulin (0.75 U/kg body weight). After insulin injection, the p.Arg457Gln mutant pigs had lower blood glucose levels than the wild-type ones all the time. Pigs are one year old. Oil-red-O staining of lipid droplet deposition, which is differentiated from white adipocytes tissue precursor cells deriving from 14-day-old piglets, Each variant and wild type were


Introduction
Type 2 diabetes associated with insulin resistance and/or pancreatic beta cell dysfunction accounts for more than 90% of diabetes cases 1 . Many studies [2][3][4][5] have shown that obesity is one of the most important risk factors for type 2 diabetes. However, some people with obesity are metabolically healthy. The capacity of subcutaneous adipose tissue to expand and store excess lipids is thought to contribute to the variation in BMI among people who develop type 2 diabetes. Recent genetic studies 6 have identi ed various favourable adiposity alleles which are associated with higher BMI, higher subcutaneous to visceral adipose tissue, higher insulin sensitivity, yet lower type 2 diabetes risk.
The minor allele of rs373863828 (p.Arg457Gln), found uniquely in populations from the islands of the Paci c, encoding cAMP-responsive element binding protein 3 (CREB3) factor regulatory (CREBRF) is associated with the largest increase in BMI reported for a common gene variant (approximately 1.4kg/m2 per allele) yet intriguingly, 1.6-fold decrease in the risk of type 2 diabetes [7][8][9][10][11] . CREBRF, expressed widely in a variety of tissues, is a speci c negative regulator of CREB3; it can translocate nuclear CREB3 out of the nucleus and promote CREB3 protein degradation, which regulates the CREB3/Luman protein localized at the endoplasmic reticulum CREBRF (CREB3 regulatory factor) 12 . Furthermore, all CREB3 family submembers play a role in unfolded protein response 12,13 . CREBRF is a potent tumor suppressor of glioblastoma that functions by blocking hypoxia-induced autophagy and is also involved in the regulation of angiogenesis by high-density lipoproteins [14][15][16] . While the CREB3/Luman protein was identi ed through its association with herpes simplex virus-related host cell factor 1, indicating that this protein may play a role in virus emergence from latency 17 , its mechanistic effects on obesity or type 2 diabetes is unclear.
Appropriate animal models are necessary to de ne the function of the CREBRF in relation to obesity and mechanisms for protection from T2DM. To date, most studies of CREBRF p.Arg457Gln are limited to the description of phenotypes among speci c human populations [18][19][20][21][22][23][24] . The CREBRF knock-out mice has been used to examine anxiety-like behavior and circulating glucocorticoids in response to various acute stress conditions 25,26 . Orthologous mouse CREBRF p.Arg458Gln variant gene knock-in mice showed increase in length at 8 weeks post birth, but showed no signi cant difference in body mass or fasting glucose or insulin at 10 weeks 21 Compared with rodents, the anatomy, physiology, size of organ, and life expectancy of pigs are more similar to humans 27,28 . The circadian rhythm (diurnal), eating behavior (discontinuously during the day), fat composition and deposition, gastrointestinal structure, islet structure, beta cell mass, insulin, and endocrine cell type proportion of pigs are similar to humans [29][30][31][32][33][34][35][36][37] . Pigs may be more suitable animal models for studying the relationship between obesity and type 2 diabetes among different genotypes 38 .
Here, we generated a CREBRF missense variant pig model with p.Arg457Gln mutations anchored in exon 7 via the CRISPR/Cas9 system. CREBRF p.Arg457Gln pigs fed with normal chow diet have elevated fat deposition and insulin sensitivity, thereby authentically replicating the phenotypes of human population with p.Arg457Gln mutations. The obesity of pigs with the CREBRF p.Arg457Gln variant was caused by increased number rather than size of adipose tissues. Moreover, the reduced risk of obesity-induced type 2 diabetes was found to be related to the lower reactive oxygen species in the adipose tissue, which would prevent insulin resistance. This study provides a new perspective for the metabolically healthy adiposity resulting from CREBRF p.Arg457Gln.

Results
Generation of CREBRF p.Arg457Gln pigs and germline transmission of the variant A CRISPR/Cas9-mediated, HR-dependent integration strategy was used to introduce the corresponding human mutation p.Arg457Gln (c.1370G>A) into porcine endogenous CREBRF locus. First, we compared the protein sequence and coding sequence of human CREBRF with that of pigs and found 99% and 94% homology, respectively. The homologous site of human missense mutation (c.1370G>A) in pig is also c.1370G>A; thus, we designed sgRNA and ssODNs with 89 nt containing c.1370G>A as homology-directed repair (HDR) donor template to introduce the missense mutation (c.1370G>A) into the pig CREBRF gene ( Figure 1A). Similar sense mutations were introduced in the ssODNs at the single-guide RNA (sgRNA) target site to avoid retargeting (Fig. 1a).
The Cas9-gRNA plasmid and CREBRF c.1370G>A ssODNA were co-transfected into pig fetal broblast cells (PFFs) derived from 35-day-old fetus through electroporation. After selection using G418 for 7-9 days, 370 cell clones were selected and expanded. The sequencing of the PCR products of the objective DNA fragment con rmed that 14 of 370 cell colonies were modi ed with the biallelic mutation (CREBRF c.1370G>A/ p.1370G>A ). Cell colony nos. 49,75,289,327, and 358 were used as donor cells for somatic cell nuclear transfer (SCNT) (Fig. 1e). A total of 843 cloned embryos were transferred into the oviducts of four recipient surrogates. Three of them were pregnant and gave birth to 12 piglets including 7 females and 5 males (Fig. 1c, 1d and 1e). The genotyping results indicated that all 12 cloned piglets were homozygous for the mutated CREBRF gene (CREBRF c.1370G>A/ c.1370G>A , Fig. 1b). Two CREBRF p.Arg457Gln variant female founders were mated to two wild-type (WT) Bama male pigs and gave birth to 24 rst-generation (F1) offspring. PCR genotyping (Fig. 1b) revealed that 17 of the 24 F1 pigs carried the CREBRF variant including 5 female homozygotes, 2 male homozygotes, 7 male heterozygotes, and 3 female heterozygotes (Fig. 1e). These results validate the feasibility of germline transmission of CRISPR/Cas9based genetic engineering in pigs and indicate that the CREBRF p.Arg457Gln variant might not affect fertility.

CREBRF p.Arg457Gln variant pigs displayed obesity and lower insulin resistance
In humans, the CREBRF p.Arg457Gln variant is inherited in an autosomal-dominant pattern and is known to strongly associate with body mass index (BMI), including body fat percentage, abdominal circumference, and height 7,8 . Hence, we then characterized the metabolic phenotype of the CREBRF p.Arg457Gln variant pigs. As predicted, the body weight of CREBRF p.Arg457Gln variant pigs signi cantly increased compared with that of the control (Fig. 2a) (WT, **vs. ** p<0.05). Body length and abdominal circumference were elevated in CREBRF p.Arg457Gln pigs ( Fig. 2b and 2c) (**vs. ** p<0.05, **vs. ** p<0.05 ). Computer tomography (CT) scan-based body composition analysis revealed that the majority of the body mass difference between the CREBRF p.Arg457Gln variant and wildtype pigs was attributed to fat deposition when fed on a chow diet (Fig. 2d). The mRNA expression of CREBRF was not negatively affected ( Fig. 2e), while protein expression was not changed in subcutaneous fat tissues of homozygous CREBRFc.1370G>A/ c.1370G>A pigs (Fig. 2f). After dissection, white adipose tissue (WAT) appeared thicker in the CREBRF p.Arg457Gln variant pigs (data not show). Histological analysis revealed smaller but more abundant lipid accumulation in the subcutaneous adipose tissue (SAT) of the CREBRF p.Arg457Gln variant pigs than that in the control ( Fig. 2g and 2h)". Similar to humans, CREBRF p.Arg457Gln pigs also displayed lower fasting glucose and insulin levels. Glucose tolerance evaluated by intravenous glucose tolerance test (IGTT) (Fig. 2i) revealed that the blood glucose level increased rapidly in CREBRF p.Arg457Gln variant pigs and wild-type pigs shortly after glucose injection but it returned to the basal level faster in variant pigs than in WT ones. The insulin tolerance test (ITT) showed that the blood glucose level reduced more quickly and maintained low for longer time in variant pigs after insulin injection (Fig. 2j) than that in WT counterpart. These data indicate that CREBRF p.Arg457Gln variant pigs were predisposed to obesity and might have improved insulin sensitivity, consistent to the phenotypic correlation in CREBRF p.Arg457Gln variant in humans.

CREBRF p.Arg457Gln enhanced preadipocyte differentiation in WAT
The morphology and size of adipocytes were examined in the SAT of 10-weekold CREBRF p.Arg457Gln variant and wild-type pigs to further investigate the obesity phenotype.
Histological analysis and hematoxylin-eosin staining ( Fig. 2g and 2h) showed that adipocyte size was obviously smaller in the SAT of CREBRF p.Arg457Gln variant pigs accompanied with increased adipocyte number. We also examined the phenotype of adipocytes through in vitro culture. We isolated the precursor adipocytes from subcutaneous adipose tissues and differentiated them into mature adipocytes. The differentiation method was modi ed from Zheng 39 , and the transformation rate was close to 90%. As predicted, the precursor adipocytes from CREBRF p.Arg457Gln variant pigs produced more mature adipocytes in vitro than from the wild-type littermates ( Fig. 2k and 2l). This nding suggests that CREBRF p.Arg457Gln promotes the differentiation of precursor adipocytes.
We explored the molecular basis of CREBRF p.Arg457Gln-mediated fat deposition in SAT. A global comparison of the mRNA expression pro le of the CREBRF p.Arg457Gln variant and wild-type pigs was performed ( Fig. 3). At a threshold of 1.5-fold change, RNA sequencing (RNA-seq) analysis identi ed 667 signi cantly up-regulated genes and 1126 signi cantly down-regulated genes in the CREBRF p.Arg457Gln variant relative to the wild type (Fig. 3a). These differently expressed genes were found to be involved in multiple gene ontology (GO) terms, such as cell proliferation, energy metabolism, ion response, and fat metabolism. Pathway enrichment analysis markedly focused on the PPAR signaling pathway (Fig. 3b) and showed that the mRNA expression levels of several genes related to adipocyte differentiation (ANGPTL4, SORBS1, ACDC, PLIN2, PLIN4, and PLIN5) were signi cantly up-regulated in the CREBRF p.Arg457Gln variant pigs (P < 0.01) (Fig. 3c). We observed that fatty acid synthesis-related genes strongly preferred to present in the down-regulated gene regions (P < 0.01), such as SCD, ACAT2, FASN, ECHS1, ELOVL6, FADS1, FADS2, and TECR. These ndings were also con rmed by quantitative PCR (qPCR) test (Fig. 3d).
Overall, CREBRF p.Arg457Gln regulated fat deposition probably by promoting adipocyte differentiation, rather than accelerating the de novo lipogenesis of adipocyte, which result in increased adipocyte number instead of expanding its size. CREBRF p.Arg457Gln improved insulin sensitivity through repression of oxidative capacity CREBRF p.Arg457Gln variant pigs exhibited elevated insulin sensitivity. To further identify changes in the global gene expression in CREBRF p.Arg457Gln variant pigs that might account for the observed improvement of glucose uptake, we performed a Gene Set Enrichment Analysis (GSEA) on genes that were signi cantly different between CREBRF p.Arg457Gln pigs and wild-type ones (Fig. 3b). Gene sets including tricarboxylic acid cycle (TAC) ( Fig. 3e and 3g) and oxidative phosphorylation (OXPHOS) ( Fig. 3f and 3h) were found to be strongly enriched in the up-regulated but not in the down regulated pool. The plot of the position of each of the 132 genes in the oxidative phosphorylation set showed that the downregulated genes (87) of oxidative phosphorylation was almost two times more than the up-regulated ones (43). This nding strongly argues that the enrichment of the oxidative phosphorylation set genes in the up-regulated pool is not simply a consequence of the most abundant oxygen metabolism-related genes Page 7/28 detected by the RNA-seq, which is a phenotype caused by the CREBRF missense mutation. Metabolome analysis revealed the accumulation of the intermediates of glycolysis (Fig. 4a, 4b and 4c), including phosphoenolpyruvate (PEP) in CREBRF p.Arg457Gln variant pigs; meanwhile, glycerone phosphate, N-Acetyl-D-glucosamine 6-phosphate, glutathione disul de, 1,2-Diacylglycerol-Bile-PC-pool, Phosphodimethylethanolamine, and D-Ribose 5-phosphate were reduced 40,41 . Hence, pigs rendered with the CREBRF p.Arg457Gln variant may probably had lower SAT oxidative capacity .
Excessive production of peroxides in the electron transport chain of mitochondria will increase the number of reactive oxygen species (ROS) and trigger a state of oxidative stress, which could lead to insulin resistance and even type 2 diabetes 42,43 . Repression of oxidative capacity in SAT from CREBRF p.Arg457Gln variant pigs suggests that the reduction of ROS may contribute to the improved insulin sensitivity. To test this idea, we measured the ROS production of SAT from CREBRF p.Arg457Gln and wild-type pigs with a Dihydroethidium (DHE) probe. The CREBRF p.Arg457Gln variant pigs displayed signi cantly lower ROS than the wild-type pigs (Fig. 5a). We then detected lipid peroxide content in the SAT from CREBRF p.Arg457Gln variant and wild-type pigs. The levels of 4-HNE and MDA evidently decreased in the CREBRF p.Arg457Gln variant pigs ( Fig. 5b and 5c). Antioxidant enzyme activity was measured. We observed increased glutathione peroxidase (GPX), superoxide dismutase (SOD), and catalase (CAT) activity ( Fig. 5d, 5e, 5f and 5g). Correspondingly, the mRNA expression levels of Gpx1, Sod2, and Prdx5 increased. Thus, the transcriptome and metabolome data indicate that the CREBRF p.Arg457Gln variant reduced the oxidative metabolism in SAT.

Discussion
In this study, we generated and characterized a pig model with missense CREBRF p.Arg457Gln variant by combining CRISPR/Cas9 editing system with somatic cell nuclear transfer. CREBRF p.Arg457Gln pigs fed with normal chow diet have elevated fat deposition and insulin sensitivity at around 20 weeks old, authentically replicating the increased BMI yet reduced risk of type 2 diabetes phenotypes observed in human populations with p.Arg457Gln mutations.
We obtained 12 homozygous founder pigs with females and males. Seventeen F1 pigs were identi ed as CREBRF p.Arg457Gln variant carriers with heterogeneous and homozygous mutations. All the CREBRF p.Arg457Gln variant carriers, regardless of heterozygote or homozygote, male or female, displayed obviously increased body weight, abdominal circumference, and body length compared with the wild-type littermates. All these phenotypes were resulted from normal diet chow, similar to the human population with the same genotype 19,[21][22][23] . The model is different from obesity linked to diabetes models, such as over-expressing fat mass and obesity-associated (FTO) mice 44 , mice with genetic deletions of melanocortin 4 receptor(MC4R) 45 leptin-de cient mice 46 and zebra sh obesity models 47 , in which the obesity phenotype was primarily caused by increased appetite and food intake and decreased physical activity 48 . The CREBRF p.Arg457Gln pigs could store more fat without overeating. Moreover, mutations in the FTO gene often lead to the development of abnormalities [49][50][51] , which was not seen among CREBRF p.Arg457Gln pigs 42 .
According to the location of deposition, fat can be divided into visceral fat and subcutaneous fat. In the CREBRF p.Arg457Gln variant pigs, body weight gain could be ascribed to increased fat deposition mainly in subcutaneous fat given that we did not observed distinct fat accumulation in the visceral organ (data not shown). The CT results also con rmed limited visceral but mainly increaced subcutaneous fat deposition in CREBRF p.Arg457Gln variant pigs. The metabolically unhealthy obesity phenotype is associated with enlarged adipocytes with reduced buffering capacity for lipid storage, thereby exposing organs, such as liver, skeletal muscle, pancreas, the heart, and kidneys, to an excessive in ux of lipids, leading to ectopic fat deposition and insulin resistance 52,53 . Ectopic lipid deposition is deleterious [54][55][56] , while deep abdominal subcutaneous adipose tissues seem to be "metabolically healthy," and were proven to have higher expression of pro-in ammatory, lipogenic, and lipolytic genes and contain higher proportions of saturated fatty acids and small adipocytes 57,58 . Increased capacity for subcutaneous fat deposition in the CREBRF p.Arg457Gln variant pigs suggests that this obesity may confer lower risk for type 2 diabetes.
The insulin resistance of adipocytes is recognized as one of the hallmarks of type 2 diabetes pathogenesis 59 . IGTT and ITT data indicated that CREBRF p.Arg457Gln variant pigs have improved insulin sensitivity, consistent with the phenotypic correlation in humans with the CREBRF p.Arg457Gln variant. Previous reports showed that excessive free fatty acid (FFA) in circulation is related to obesity, insulin resistance, and type 2 diabetes. Adipocytes can e ciently sequester excessive glucose and fatty acids from the circulation and store them in intracellular lipid droplets 60 . When an individual becomes obese, adipocytes expand and become hypertrophic with intracellular lipid accumulation; they eventually rupture and fatty acids ow out [61][62][63][64] , thereby inhibiting insulin-stimulated glucose transport or phosphorylation, and glycogen synthase activity 65,66 . The adipocytes of SAT in CREBRF p.Arg457Gln variant pigs had smaller size but increased number, suggesting that the CREBRF p.Arg457Gln variant increases the storage capacity of adipose tissue by promoting the differentiation of adipocyte progenitor cells into more adipocytes (hyperplasia) instead of expanding their size (hypertrophy). The RNA-seq data also con rmed that adipogenic differentiation associated peroxisome proliferator-activated receptor (PPAR) signaling pathway was signi cantly enriched and fatty acid transportation-related genes were obviously down-regulated in the CREBRF p.Arg457Gln variant pigs. We also validated adipocyte hyperplasia through examination of the differentiation capacity of preadipocyte cells in vitro. More new adipocytes were generated from the same number of CREBRF p.Arg457Gln preadipocytes than the wild-type ones. Therefore, pigs with the CREBRF p.Arg457Gln variant contain numerous and relatively smaller adipocytes, which improve their energy storage capability before enlarging and risking toxic ectopic lipid accumulation related rupture of hypertrophic adipocytes and subsequent increased plasma FFA level and insulin resistance. Palmitoleic acid, which has bene cial effects on glucose homeostasis in dietary obesity and a potential target for development of diabetes drugs 67 , was greatly enhanced in the CREBRF p.Arg457Gln variant pigs. This nding also suggests the important role of the CREBRF p.Arg457Gln variant in type 2 diabetes protection.
Increasing lines of evidence in experimental and clinical studies suggest that obesity causes redox imbalance in fat tissues and produces excessive ROS and harmful free radicals, which result in oxidative stress. Oxidative stress is thought to contribute to diabetes progression by inducing insulin resistance [68][69][70][71][72] . In the present study, several mechanisms by which the CREBRF p.Arg457Gln variant could attenuate adipocytes oxidative stress were identi ed, including down-regulating OXPHOS and reducing ROS production. The particular reaction of ROS with lipids is generally known as "lipid peroxidation.  74,75 . The mRNA expression levels of Gpx1, Sod2, and Prdx5 increased, indicating that the activities of antioxidant enzymes were enhanced, which would detoxify ROS, prevent the harmful effects of active oxidizing reaction, and maintain redox homeostasis. Therefore, there was lower risk of obesity-induced type 2 diabetes in pigs with the CREBRF p.Arg457Gln variant.
In summary, these pig studies demonstrate that p.Arg457Gln variant in CREBRF promotes the differentiation of precursor adipocytes into more mature adipocytes for storing more energy by increasing their number (hyperplasia) rather than their size (hypertrophy). Secondly, the p.Arg457Gln variant in CREBRF reduces oxidative capacity and increases the antioxidative ability of SAT, thereby scavenging excess ROS. Both effects of the CREBRF p.Arg457Gln variant reduce insulin resistance and reduce the risk of type 2 diabetes (Fig. 6).

Ethics Statement and Animal Housing
All experiments involving animals were conducted according to the guidelines for the care and use of laboratory animals established by the Guangdong Association for Laboratory Animal Science and approved by the Animal Ethics Committee of the Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences. Pigs were raised at the Jiangmen Farm Animal Research Center. Feed was given twice daily without limit.

Plasmids
Given that Arg457 in the pig CREBRF protein was identi ed as homologous to human Arg457 that is mutated in Samoans, we planned to introduce a point mutation homologous to human p.Arg457Gln on exon 7 of the pig CREBRF gene by using CRISPR/Cas9-based genome editing. The sequences are listed in Supplementary Table 1. The pig endogenous CREBRF fragment was cloned into the U6-gRNA vector along with the annealed sgRNA oligos, which were designed using the tool available at the website http://crispr.mit.edu, to construct a targeting vector. Additionally, a donor oligonucleotide was used to mutate CGA encoding Arg457 to CAA (encoding Gln) by homology-directed repair. The CRISPR/Cas9 construct was obtained as previously described.

Cell Culture and Transfection
Primary pig PFFs were isolated as previously reported 76 . Targeting vector CRISPR/Cas9 construct were co-injected with the donor oligonucleotide into cultured PFF cells by electrotransfection. Forty-eight hours after transfection, cells were harvested using 0.25% trypsin/EDTA (Gibco), and the cell density was calculated using a handheld automated cell counter (Millipore). Single cells were plated in each well of 96-well plates by limiting dilution and cultured for ∼10 d in the cell culture medium supplemented with 2.5 ng/mL basic broblast growth factor (Sigma). The medium was replaced every 3 days. Con uent cell colonies were propagated and genotyped by PCR and sequencing.

Nuclear Transfer and Embryo Transfer
Pig ovaries were collected from a local slaughterhouse and transported to the laboratory within 1 h in 0.9% saline maintained at 37 °C. The in vitro maturation of oocytes, enucleation, microinjection, and the fusion of reconstructed oocytes were performed in our laboratory according to previously described methods. The reconstructed embryos were cultured in porcine zygote medium in 5% CO 2 at 39 °C for 14-16 h until embryo transfer. Embryos in good condition were surgically transferred into the oviduct of a surrogate the day after observed estrus. Gestation was detected by ultrasound at day 28 after embryo transfer. All of the cloned piglets were delivered by natural birth.

RNA Preparation and qPCR Analysis
Total RNA was isolated from pig SAT with TRIzol (Invitrogen). First-strand cDNA synthesis was performed using a FastQuant RT Kit (Tiangen Biotech). Quantitative real-time PCR reactions were performed using SYBR Premix Ex Taq (Tli RNaseH Plus; Takara) on an Agilent Mx3005p (Agilent Technologies) with reaction volume of 20 μL. The primer sequences are listed in Supplementary Table 2. Relative gene expression was calculated using comparative cycle threshold (2−ΔΔCt) method. Statistical analysis was performed with GraphPad Prism 8.0.

Western Blot Analysis
SAT were dissected, frozen immediately in liquid nitrogen, and stored at -80 °C until use. Total proteins from tissue or cultured adipocyte were extracted using the Minute Total Protein Extraction Kit (Invent Biotechnologies, Inc.). The proteins were subjected to Western blot analysis with the following antibodies: anti-CREBRF (PA5-68552, 1:1000; Invitrogen, Inc.), and anti-actin (AF7018,1:2000, A nity). The blots were developed using HRP-conjugated secondary antibodies and an ECL Plus system. All signals were visualized and analyzed with a Tanon 5200 (Tanon Science & Technology Co. Ltd.)

Computed tomography (CT)
Ten-week-old female Bama pigs of the CREBRF R457R variant and wild type were used for the experiments. All pigs were clinically healthy based on physical examination. CT was conducted using a helical CT scanner with a single-detector CT (GE Medical System, USA) beginning at the upper edge of the liver and continuing caudally to the L4 level. The pigs were placed under general anesthesia for the procedure. Image acquisition parameters included a matrix of 512 × 512, a large-scan eld of view, a 1.25 mm slice thickness, 120 kVp, 60 mA, and a pitch of 1.3. CT scans were conducted to determine the distribution of abdominal fat identi ed based on the number measured in Houns eld Units (HU). CT number ranges of visceral and subcutaneous abdominal adipose tissue were obtained by manually setting the region of interests (ROIs) corresponding to each of the images obtained at the L3, L4 levels by three radiologists. In this procedure, we de ned the attenuation range of fat tissue from -190 HU to -32 HU. By adjusting the fat ranges, we easily obtained the areas of total abdominal fat by using the built-in software for the helical CT scanner. We then compared the overall abdominal fat mass with the fat areas calculated at the L3 and L4 levels by de ning them as landmarks to measure the total abdominal fat distribution.

Histological Analysis
Adipose tissues were xed in 4% paraformaldehyde/1× PBS overnight at 4 °C and dehydrated through sequential ethanol washes. The tissue was embedded in para n before sectioning and then stained with hematoxylin and eosin. The stained sections were visualized and photographed under bright-eld microscopy.

Glucose Tolerance Test
For intravenous glucose tolerance tests, pigs were fasted for 16 h and challenged with intravenous injection of 50% glucose (1.2 mL/kg body weight). For insulin tolerance tests, pigs were fasted for 12 h and then given an intraperitoneal injection of insulin (0.75 U/kg body weight). Blood glucose levels were measured by using a glucose monitor (Accu-Chek. Performa, Roche, Germany) in blood drawn from the auricular region at 0, 5, 10, 15, 30, 45, 60, 90, and 120 min.

Transcriptome Pro ling
Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase-free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo(dT) beads. The enriched mRNA was fragmented into short fragments by using fragmentation buffer and reverse transcribed into cDNA with random primers. Second-strand cDNA was synthesized by DNA polymerase I, RNase H, dNTP, and buffer. The cDNA fragments were puri ed with QiaQuick PCR extraction kit (Qiagen, Venlo, The Netherlands), end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR ampli ed, and sequenced using Illumina HiSeq2500 by Gene Denovo Biotechnology Co. (Guangzhou, China). Raw reads were mapped against the reference genome by using HISAT2. 2.4 with "-rna-strandness RF". The mapped reads were converted to FPKM (fragments per kilobase of exon per million fragments mapped) by running StringTie software to determine gene expression. RNA differential expression was analyzed by DESeq2 software between two different groups (and by edgeR between two samples). The genes/transcripts with the parameter of false discovery rate (FDR) below 0.05 and absolute fold change≥2 were considered differentially expressed genes/transcripts. Gene set enrichment analysis was performed using software GSEA and MSigDB to identify whether a set of genes in speci c GO terms/pathways/DO terms shows signi cant differences in the two groups. Brie y, we input gene expression matrix and rank genes by SinaltoNoise normalization method. Enrichment scores and p value were calculated in default parameters.

Metabolome analysis
About 50 mg of the sample was weighed to an EP tube. After the addition of 1000 μL of extract solvent (acetonitrile-methanol-water, 2:2:1, containing internal standard), the samples were vortexed for 30 s, homogenized at 45 Hz for 4 min, and sonicated for 5 min in ice-water bath. The homogenization and sonication were repeated three times, followed by incubation at -20 °C for 1 h and centrifugation at 12000 rpm and 4 °C for 15 min. The resulting supernatants were transferred to LC-MS vials and stored at -80 °C until the UHPLC-QE Orbitrap/MS analysis. The quality control (QC) sample was prepared by mixing an equal aliquot of the supernatants. LC-MS/MS analyses were performed using an UHPLC system (1290, Agilent Technologies) with a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) coupled to Q Exactive (Orbitrap MS, Thermo). The mobile phase A was 0.1% formic acid in water for positive, and 5 mmol/L ammonium acetate in water for negative, and the mobile phase B was acetonitrile. The QE mass spectrometer was used to acquire MS/MS spectra on an information-dependent basis (IDA) during the LC/MS experiment. MS raw data les were converted into the mzML format by using ProteoWizard and processed by R package XCMS (version 3.2), including retention time alignment, peak detection, and peak matching. OSI-SMMS (version 1.0, Dalian Chem Data Solution Information Technology Co. Ltd.) was used for peak annotation after data processing with the in-house MS/MS database. For preliminary visualization of differences between different groups of samples, the unsupervised dimensionality reduction method principal component analysis (PCA) was applied using R package models (http://www.r-project.org/). PLS-DA, a supervised dimensionality reduction method in which class memberships are coded in matrix form into Y to better distinguish the metabolomics pro le of two groups by screening variables correlated to class memberships, was applied to compare the groups by using R package models (http://www.r-project.org/). A variable importance in projection (VIP) score of (O)PLS model was applied to rank the metabolites that best distinguished between two groups. The threshold of VIP was set to 1. In addition, T-test was used as a univariate analysis for screening differential metabolites. Those with a P value of T test<0.05 and VIP≥1 were considered differential metabolites between two groups.

Porcine Primary Preadipocyte Isolation and in Vitro Differentiation
Adipose tissues were harvested from 2-week-old piglets, minced, and digested with 2 mg/mL collagenase type I (Sigma) in DMEM/F12 containing 1% fatty acidfree BSA (Sigma) for 60 min at 37 °C. Stromal vascular fraction cells were collected with a cell strainer (70 μm diameter), plated, and grown in DMEM/F12 (Gibco) supplemented with 10% FBS (Sigma) and 1% penicillin-streptomycin. For white adipocyte differentiation, cells were grown to con uence and treated with human WAT induction medium

ROS Assay
ROS was detected using DHE staining. DHE is a cell-permeable uorogenic probe that reacts with ROS to form ethidium and emits red uorescence. Adipose tissues were weighed and homogenized, and then homogenate supernatant was taken, mixed with DHE probe, and incubated in the dark for 30 min at 37 ℃. Fluorescence intensity was detected at the excitation wavelength of 488-535 nm and emission wavelength of 610 nm by using an inverted uorescence microscope (DP72/CKX41, Olympus). Finally, the reactive oxygen species level of tissues was expressed by uorescence intensity/mg protein.

4-HNE and MDA Analysis
4-HNE and MDA was measured by enzyme-linked immunosorbent assay and TBA uorescence method, respectively. First, adipose tissues were weighed and homogenized, then 4-HNE was detected according to the manufacturer's instruction. First, set blank hole, standard hole, sample hole. Add sample 50 μL accurately to the Elisa-coated plate, add sample dilution 40 μL to the test sample well, then add sample 10 μL to the test well. Add the sample to the bottom of the enzyme plate well and mix gently. Then, after sealing the plate with sealing plate membrane, incubate at 37℃ for 30 minutes. Uncover sealing plate membrane, discard liquid, dry by swing, add washing solution to each well, rest for 30 seconds and then drain, repeat 5 times. Add HRP-conjugate reagent 50 μL to each well, except blank well. Incubate at 37℃ for 30 minutes and wash. Afterwards, add chromogenic agent A 50 μL and chromogenic agent B 50 μL to each well, mix gently. Add stop solution 50 μL to each well at 37℃ for 10 min away from light, stop the reaction. Finally, the absorbance of each hole was measured in sequence at 450 nm wavelength with blank air conditioning zero. MDA was detected by mixed TBA dye, and uorescence intensity was detected at the absorbance wavelength of 532 nm by using a microplate reader. GPX, SOD, and CAT Assay GPX, SOD, and CAT assays were conducted using exclusive kits according to the manufacturer's instructions (H545-1, A001-3-1 andA007-1-1; Nanjing Jiancheng Bioengineering Institute). For detail operation steps, please refer to the commercial manual.
Oil Red Staining of Mature Adipocytes Adipocytes in one well of the six-well plate were used as an example. The cells were gently washed twice with PBS and xed with 500 µL of 4% paraformaldehyde for 30 min. Oil red working liquid was prepared: Distilled water = 3:2, mix and lter through a 70 µM lter. After the cells were xed, they were washed with 500 µL 60% isopropanol, excess water was removed, and the cells were turned upside down until they were completely dried. The sample was added with 300 μL of oil red working solution and stained for 10 min without shaking. The oil red working uid was absorbed, and the cells were gently washed with DDH 2 O for 4 times. After washing, the supernatant was absorbed and discarded. The sample was placed under a microscope, and photos were taken. For further absorbance test, the excess DDH 2 O should be completely absorbed and dried. The sample was added with 750 μL of 100% isopropyl alcohol and incubated for at least 10 min. Gentle blowing was conducted to ensure that all the oil red staining solution was dissolved in isopropyl alcohol. The dye solution was transferred to a 1.5mL centrifuge tube. A new 1.5 mL centrifuge tube lled with 100% isopropyl alcohol of the same volume as the control was prepared. OD value was obtained at the wavelength of 500 nm and read in 0.5 seconds.

Statistical Analysis
The statistical data reported include results from at least three biological replicates. All results are expressed as mean ± SEM. For normal distribution data, differences between two groups were assessed by unpaired Student's t tests.
Declarations Figure 1 CRISPR/Cas9-mediated, HR-independent integration e ciently produced CREBRF p.Arg457Gln pigs and they generated inheritable offsprings. a Schematic representation of the targeting vector. Exons of CREBRF are shown as black boxes, and the orange box in exon 7 represents the sgRNA targeting site, which is enlarged to a rectangle box with the targeting sequence in the red box, the mutated bases in red, and the synonym mutations in capital letters. b Sequencing PCR products of objective DNA fragment con rmed CREBRFc.1370G>A, p.457Arg>Gln insertion of donor DNA in the cloned pigs. The top one is homozygous CREBRFc.1370G>A, p.457Arg>Gln mutant genotype and the bottom one is heterozygous CREBRFc.1370G>A, p.457Arg>Gln mutant genotype with the bimodal sites being a synonym mutation at the target sites. The red arrow points to the mutation site. c Eight months old wild type(left) and CREBRF p.Arg457Gln mutant F0 pig(right). The CREBRF p.Arg457Gln mutant pig is visibly obese that its belly drags the oor. d The rst-generation (F1) pigs (left) at day 15 and their fourteen months old mother pig (CREBRF p.Arg457Gln, F0, right). e CRISPR/Cas9 and ssODN-mediated point mutation in porcine fetal broblast cells and generation of CREBRF p.Arg457Gln F0 and F1 p signi cant differences compared with controls are indicated by **P < 0.001,**P < 0.01 and*P < 0.05. Data were analyzed with a two-sample t test. OXPHOX: Oxidative phosphorylation. GSEA: Gene set enrichment analysis. Each the p.Arg457Gln variant group and wildtype group were clustered using four healthy adult pigs(more than one year old), respectively. Signi cant difference was assessed by p<0.05, FDR-adjusted.

Figure 4
Identi cation of differential metabolites in subcutaneous fat between p.Arg457Gln variant pigs and wildtype pigs by untargeted metabolome analysis. a Heatmap of the contents of differential metabolites.
b Top 20 KEGG enrichment of the differential metabolites. c Cluster analysis of differential metabolites related to Glycolysis, Lipogenesis and Pre-oxidative metabolic processes. P 0.05 and VIP≥1 were considered differential metabolites between these two groups.

Figure 5
Detection of lipid peroxidation level in subcutaneous fat between p.Arg457Gln variant pigs and wildtype pigs. a ROS production. b 4-HNE content. c MDA content. d GPX activity. e SOD activity. f CAT activity. g RNA expression level of Prdx5, Sod2 and Gpx1. Data are mean ± sem of biologically independent samples. *P<0.05; **P<0.01;***P<0.001.

Figure 6
Illustration summarizing the metabolism-related role of CREBRF-p.Arg457Gln variant in the mitochondria of adipocytes under obese conditions. CREBRF p.Arg457Gln variant promotes the generation of adipocyte to increase energy storage and reduces the production of ROS by reducing oxidation capacity, which protects the body from lipid peroxidation and reduces insulin resistance.

Supplementary Files
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