Emerging roles of MRG15 in liver metabolic diseases.

MORF4 (mortality factor on chromosome 4)-related gene 15 (MRG15) is a chromodomain protein that exists in various multiprotein complexes involved in transcription, DNA repair, and development. Here we summarize the recent advances involving MRG15 in modulating liver metabolism, both through its chromatin-binding capability and independently of it, highlighting MRG15 as a potential therapeutic target for liver metabolic diseases.

Structural and functional insights into the epigenetic regulator MRG15.

MORF4-related gene on chromosome 15 (MRG15), a chromatin remodeller, is evolutionally conserved and ubiquitously expressed in mammalian tissues and cells. MRG15 plays vital regulatory roles in DNA damage repair, cell proliferation and division, cellular senescence and apoptosis by regulating both gene activation and gene repression via associations with specific histone acetyltransferase and histone deacetylase complexes. Recently, MRG15 has also been shown to rhythmically regulate hepatic lipid metabolism and suppress carcinoma progression. The unique N-terminal chromodomain and C-terminal MRG domain in MRG15 synergistically regulate its interaction with different cofactors, affecting its functions in various cell types. Thus, how MRG15 elaborately regulates target gene expression and performs diverse functions in different cellular contexts is worth investigating. In this review, we provide an in-depth discussion of how MRG15 controls multiple physiological and pathological processes.

BACH1 controls hepatic insulin signaling and glucose homeostasis in mice.

Hepatic insulin resistance is central to the metabolic syndrome. Here we investigate the role of BTB and CNC homology 1 (BACH1) in hepatic insulin signaling. BACH1 is elevated in the hepatocytes of individuals with obesity and patients with non-alcoholic fatty liver disease (NAFLD). Hepatocyte-specific Bach1 deletion in male mice on a high-fat diet (HFD) ameliorates hyperglycemia and insulin resistance, improves glucose homeostasis, and protects against steatosis, whereas hepatic overexpression of Bach1 in male mice leads to the opposite phenotype. BACH1 directly interacts with the protein-tyrosine phosphatase 1B (PTP1B) and the insulin receptor ß (IR-ß), and loss of BACH1 reduces the interaction between PTP1B and IR-ß upon insulin stimulation and enhances insulin signaling in hepatocytes. Inhibition of PTP1B significantly attenuates BACH1-mediated suppression of insulin signaling in HFD-fed male mice. Hepatic BACH1 knockdown ameliorates hyperglycemia and improves insulin sensitivity in diabetic male mice. These results demonstrate a critical function for hepatic BACH1 in the regulation of insulin signaling and glucose homeostasis.

Excessive apoptosis of Rip1-deficient T cells leads to premature aging.

In mammals, the most remarkable T cell variations with aging are the shrinking of the naïve T cell pool and the enlargement of the memory T cell pool, which are partially caused by thymic involution. However, the mechanism underlying the relationship between T-cell changes and aging remains unclear. In this study, we find that T-cell-specific Rip1 KO mice show similar age-related T cell changes and exhibit signs of accelerated aging-like phenotypes, including inflammation, multiple age-related diseases, and a shorter lifespan. Mechanistically, Rip1-deficient T cells undergo excessive apoptosis and promote chronic inflammation. Consistent with this, blocking apoptosis by co-deletion of Fadd in Rip1-deficient T cells significantly rescues lymphopenia, the imbalance between naïve and memory T cells, and aging-like phenotypes, and prolongs life span in T-cell-specific Rip1 KO mice. These results suggest that the reduction and hyperactivation of T cells can have a significant impact on organismal health and lifespan, underscoring the importance of maintaining T cell homeostasis for healthy aging and prevention or treatment of age-related diseases.

Modeling drug-induced liver injury and screening for anti-hepatofibrotic compounds using human PSC-derived organoids.

Preclinical models that can accurately predict the toxicity and efficacy of candidate drugs to human liver tissue are in urgent need. Human liver organoid (HLO) derived from human pluripotent stem cells offers a possible solution. Herein, we generated HLOs, and demonstrated the utility of these HLOs in modeling a diversity of phenotypes associated with drug-induced liver injury (DILI), including steatosis, fibrosis, and immune responses. Phenotypic changes in HLOs after treatment with tool compounds such as acetaminophen, fialuridine, methotrexate, or TAK-875 showed high concordance with human clinical data in drug safety testings. Moreover, HLOs were able to model liver fibrogenesis induced by TGFß or LPS treatment. We further devised a high-content analysis system, and established a high-throughput anti-fibrosis drug screening system using HLOs. SD208 and Imatinib were identified that can significantly suppress fibrogenesis induced by TGFß, LPS, or methotrexate. Taken together, our studies demonstrated the potential applications of HLOs in drug safety testing and anti-fibrotic drug screening.

Acute liver steatosis translationally controls the epigenetic regulator MIER1 to promote liver regeneration in a study with male mice.

The early phase lipid accumulation is essential for liver regeneration. However, whether this acute lipid accumulation can serve as signals to direct liver regeneration rather than simply providing building blocks for cell proliferation remains unclear. Through in vivo CRISPR screening, we identify MIER1 (mesoderm induction early response 1) as a key epigenetic regulator that bridges the acute lipid accumulation and cell cycle gene expression during liver regeneration in male animals. Physiologically, liver acute lipid accumulation induces the phosphorylation of EIF2S1(eukaryotic translation initiation factor 2), which consequently attenuated Mier1 translation. MIER1 downregulation in turn promotes cell cycle gene expression and regeneration through chromatin remodeling. Importantly, the lipids-EIF2S1-MIER1 pathway is impaired in animals with chronic liver steatosis; whereas MIER1 depletion significantly improves regeneration in these animals. Taken together, our studies identify an epigenetic mechanism by which the early phase lipid redistribution from adipose tissue to liver during regeneration impacts hepatocyte proliferation, and suggest a potential strategy to boost liver regeneration.

Histone phosphorylation integrates the hepatic glucagon-PKA-CREB gluconeogenesis program in response to fasting.

The glucagon-PKA signal is generally believed to control hepatic gluconeogenesis via the CREB transcription factor. Here we uncovered a distinct function of this signal in directly stimulating histone phosphorylation for gluconeogenic gene regulation in mice. In the fasting state, CREB recruited activated PKA to regions near gluconeogenic genes, where PKA phosphorylated histone H3 serine 28 (H3S28ph). H3S28ph, recognized by 14-3-3ζ, promoted recruitment of RNA polymerase II and transcriptional stimulation of gluconeogenic genes. In contrast, in the fed state, more PP2A was found near gluconeogenic genes, which counteracted PKA by dephosphorylating H3S28ph and repressing transcription. Importantly, ectopic expression of phosphomimic H3S28 efficiently restored gluconeogenic gene expression when liver PKA or CREB was depleted. These results together highlight a different functional scheme in regulating gluconeogenesis by the glucagon-PKA-CREB-H3S28ph cascade, in which the hormone signal is transmitted to chromatin for rapid and efficient gluconeogenic gene activation.

Single-cell sequencing unveils key contributions of immune cell populations in cancer-associated adipose wasting.

Adipose tissue loss seen with cancer-associated cachexia (CAC) may functionally drive cachexia development. Using single-cell transcriptomics, we unveil a large-scale comprehensive cellular census of the stromal vascular fraction of white adipose tissues from patients with or without CAC. We report depot- and disease-specific clusters and developmental trajectories of adipose progenitors and immune cells. In adipose tissues with CAC, clear pro-inflammatory transitions were discovered in adipose progenitors, macrophages and CD8+ T cells, with dramatically remodeled cell interactome among these cells, implicating a synergistic effect in promoting tissue inflammation. Remarkably, activated CD8+ T cells contributed specifically to increased IFNG expression in adipose tissues from cachexia patients, and displayed a significant pro-catabolic effect on adipocytes in vitro; whereas macrophage depletion resulted in significantly rescued adipose catabolism and alleviated cachexia in a CAC animal model. Taken together, these results unveil causative mechanisms underlying the chronical inflammation and adipose wasting in CAC.

Hepatic cytochrome P450 8B1 and cholic acid potentiate intestinal epithelial injury in colitis by suppressing intestinal stem cell renewal.

Although disrupted bile acid (BA) homeostasis is implicated in inflammatory bowel disease (IBD), the role of hepatic BA metabolism in the pathogenesis of colitis is poorly understood. Here, we found that cholic acid (CA) levels were increased in patients and mice. Cytochrome P450 8B1 (CYP8B1), which synthesizes CA, was induced in livers of colitic mice. CA-treated or liver Cyp8b1-overexpressing mice developed more severe colitis with compromised repair of the mucosal barrier, whereas Cyp8b1-knockout mice were resistant to colitis. Mechanistically, CA inhibited peroxisome proliferator-activated receptor alpha (PPARα), resulting in impeded fatty acid oxidation (FAO) and impaired Lgr5+ intestinal stem cell (ISC) renewal. A PPARα agonist restored FAO and improved Lgr5+ ISC function. Activation of the farnesoid X receptor (FXR) suppressed liver CYP8B1 expression and ameliorated colitis in mice. This study reveals a connection between the hepatic CYP8B1-CA axis and colitis via regulating intestinal epithelial regeneration, suggesting that BA-based strategies might be beneficial in IBD treatment.

MRG15 aggravates non-alcoholic steatohepatitis progression by regulating the mitochondrial proteolytic degradation of TUFM.

BACKGROUND & AIMS: How hepatic steatosis progresses to non-alcoholic steatohepatitis (NASH) is complicated and remains unclear. The mortality factor 4-like protein 1 (MORF4L1, also called MRG15) was previously identified as a master nuclear chromatin remodeler in the rhythmic regulation of lipid synthesis gene expression in the liver. Whether it also contributes to the progression from liver steatosis to NASH is unclear. METHODS: We adopted 2 different murine NASH models, liver biopsies from patients with NASH, and primary mouse and human hepatocyte cultures for functional examination of MRG15 in NASH progression. Immunoprecipitation-mass spectrometry was applied to identify protein partners of MRG15, and CRISPR targeting was used for gene depletion in liver cells in vivo. RESULTS: The MRG15 level is increased in the livers of humans and mice with NASH. The inflammatory cytokines in NASH livers stabilize MRG15 by increasing its acetylation. Considerable amounts of MRG15 associate with the outer mitochondrial membrane, where it interacts with and deacetylates the mitochondrial Tu translation elongation factor (TUFM). Deacetylated TUFM, especially at the K82 and K91 sites, is subjected to accelerated degradation by the mitochondrial ClpXP protease system. Reduced liver TUFM consequently results in impaired mitophagy, increased oxidative stress and activation of the NLRP3 inflammasome pathway. Blocking MRG15 expression protects the liver from NASH progression by increasing the stability of liver TUFM. Liver samples from patients with NASH also display a clear reduction in TUFM level, which correlates with increased MRG15 expression. CONCLUSION: Collectively, these findings uncover a mitochondrial MRG15-TUFM regulatory pathway that contributes significantly to progression from simple steatosis to NASH, and which could potentially be targeted to treat NASH. LAY SUMMARY: The incidence of non-alcoholic fatty liver disease and its progressive form non-alcoholic steatohepatitis (NASH) is increasing, posing a significant global health challenge. Herein, we have uncovered the importance of the MRG15-TUFM pathway in NASH development. This pathway is active in the mitochondria (energy powerhouse of the cell) and could be targeted for the treatment of NASH.

NEK2 promotes the migration and proliferation of ESCC via stabilization of YAP1 by phosphorylation at Thr-143.

BACKGROUND: Esophageal Squamous Cell Carcinoma (ESCC) was characterized as a regional-prevalent and aggressive tumor with high morbidity and mortality. NIMA-related kinase 2 (NEK2) is an interesting oncogene, the alteration of which leads to patients-beneficial outcomes. We aimed to explore the role of NEK2 in ESCC and excavate its mechanism. METHODS: RNA-seq data were downloaded from TCGA and GEO and analyzed by R software. The protein levels were detected by immunohistochemistry (IHC) or western blot (WB), and mRNA expression was detected by qRT-PCR. The in vitro role of proliferation and migration was detected by Transwell migration assay and by colony formation assay, respectively. The in vivo roles were explored using a subcutaneous xenograft tumor model, where immunofluorescence (IF) and IHC were employed to investigate expression and localization. The interaction between proteins was detected by immunoprecipitation. The stability of proteins was measured by WB in the presence of cycloheximide. RESULTS: A higher level of NEK2 was found in ESCC than normal esophageal epithelia in GEO, TCGA, and tissue microarray, which was associated with worse prognoses. The NEK2 knockdown impaired the proliferation and migration of ESCC, which also downregulated YAP1 and EMT markers like N-cadherin and Vimentin in vitro. On the contrary, NEK2 overexpression enhanced the migration of ESCC and elevated the levels of YAP1, N-cadherin, and Vimentin. Additionally, the overexpression of YAP1 in NEK2 knocked down ESCCs partly rescued the corresponding decrease in migration. The knockdown of NEK2 played an anti-tumor role in vivo and was accompanied by a lower level and nucleus shuffling of YAP1. In mechanism, NEK2 interacted with YAP1 and increased the stability of both endogenous and exogenous YAP1 by preventing ubiquitination. Moreover, the computer-predicted phosphorylation site of YAP1, Thr-143, reduced the ubiquitination of HA-YAP1, strengthened its stability, and thus influenced the migration in vitro. CONCLUSIONS: NEK2 is a prognostic oncogene highly expressed in ESCC and promotes the progression of ESCC in vitro and in vivo. Mechanistically, NEK2-mediated phosphorylation of YAP1 at Thr-143 protects it from proteasome degradation and might serve as a promising therapeutic target in ESCC. Video Abstract.

Targeting NECTIN-1 Based on CRISPR/Cas9 System Attenuated the Herpes Simplex Virus Infection in Human Corneal Epithelial Cells In Vitro.

PURPOSE: Viral keratitis caused by herpes simplex virus 1 (HSV-1) is a lifelong recurring disease and an unignored cause of blindness worldwide. Current antiviral therapy cannot eliminate the transcriptionally silent HSV-1 in latently infected patients. With the explosive applications of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) 9 gene-editing system in recent years, we aim to develop a CRISPR/Cas9 system targeting down the major HSV receptor, NECTIN-1 on human corneal epithelial cells (HCECs), to provide a novel strategy for herpes simplex keratitis (HSK) treatment. METHODS: The selected single guide RNAs (sgRNAs) targeting human nectin cell adhesion molecule 1 (NECTIN-1), together with Cas-9, were assembled into lentivirus. HCECs were infected with Lenti-Cas9-gRNAs to establish NECTIN-1 knockdown cells. Following HSV-green fluorescent protein (GFP) infection, cell survival and virus infection were determined by fluorescence microscopy and flow cytometry. Relative HSV DNA amount was also compared through quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Lentivirus packaged with the CRISPR/Cas9 system and the two selected sgRNAs both successfully edited down the protein levels of NECTIN-1 of HCECs. After HSV-GFP infection, the infection rate of HCECs in knockdown groups dramatically decreased, especially in the NECTIN-1 knockdown group 1. In addition, the relative HSV DNA amount of both knockdown groups was only 30% when compared with the control group. CONCLUSIONS: We successfully knocked down the NECTIN-1 expression in vitro by the CRISPR/Cas9 system, which alleviated the HSV infection in HCECs. TRANSLATIONAL RELEVANCE: This study offered a promising target for the cure of HSK.

Optimized protocols for efficient gene editing in mouse hepatocytes in vivo using CRISPR-Cas9 technology.

We provide a protocol for gene editing in mouse hepatocytes in vivo using the CRISPR-Cas9 technology via AAV delivery. This protocol describes the construction of AAV plasmids, AAV packaging, injection, and the detection of in vivo knockout efficiency. Using this protocol, we can get up to 1014 AAV and knock out genes in hepatocytes efficiently within 15 days. Moreover, we describe an optimized protocol to simultaneously target two genes via AAV delivery of CRISPR-Cas9 materials in the liver. For complete details on the use and execution of this profile, please refer to Wei et al. (2020).

Hepatic P38 Activation Modulates Systemic Metabolism Through Fgf21-Mediated Interorgan Communication.

The mechanisms underlying the pathogenesis of steatosis and insulin resistance in nonalcoholic fatty liver disease remain elusive. Increased phosphorylation of hepatic p38 has long been noticed in fatty liver; however, whether the activation of hepatic p38 is a cause or consequence of liver steatosis is unclear. Here, we demonstrate that hepatic p38 activation by MKK6 overexpression in the liver of mice induces severe liver steatosis, reduces fat mass, and elevates circulating fatty acid levels in a hepatic p38α- and FGF21-dependent manner. Mechanistically, through increasing the FGF21 production from liver, hepatic p38 activation increases the influx of fatty acids from adipose tissue to liver, leading to hepatic ectopic lipid accumulation and insulin resistance. Although hepatic p38 activation exhibits favorable effects in peripheral tissues, it impairs the hepatic FGF21 action by facilitating the ubiquitination and degradation of FGF21 receptor cofactor ß-Klotho. Consistently, we show that p38 phosphorylation and FGF21 expffression are increased, ß-Klotho protein levels are decreased in the fatty liver of either mice or patients. In conclusion, our study reveals previously undescribed effects of hepatic p38 activation on systemic metabolism and provides new insights into the roles of hepatic p38α, FGF21, and ß-Klotho in the pathogenesis of nonalcoholic fatty liver disease.

Hepatic P38 Activation Modulates Systemic Metabolism Through Fgf21-Mediated Interorgan Communication.

The mechanisms underlying the pathogenesis of steatosis and insulin resistance in nonalcoholic fatty liver disease remain elusive. Increased phosphorylation of hepatic p38 has long been noticed in fatty liver; however, whether the activation of hepatic p38 is a cause or consequence of liver steatosis is unclear. Here, we demonstrate that hepatic p38 activation by MKK6 overexpression in the liver of mice induces severe liver steatosis, reduces fat mass, and elevates circulating fatty acid levels in a hepatic p38α- and FGF21-dependent manner. Mechanistically, through increasing the FGF21 production from liver, hepatic p38 activation increases the influx of fatty acids from adipose tissue to liver, leading to hepatic ectopic lipid accumulation and insulin resistance. Although hepatic p38 activation exhibits favorable effects in peripheral tissues, it impairs the hepatic FGF21 action by facilitating the ubiquitination and degradation of FGF21 receptor cofactor ß-Klotho. Consistently, we show that p38 phosphorylation and FGF21 expffression are increased, ß-Klotho protein levels are decreased in the fatty liver of either mice or patients. In conclusion, our study reveals previously undescribed effects of hepatic p38 activation on systemic metabolism and provides new insights into the roles of hepatic p38α, FGF21, and ß-Klotho in the pathogenesis of nonalcoholic fatty liver disease.

[Applications of stem cell disease models in liver metabolic research and drug development].

The high failure rate of the new drug development has been well recognized. Relying on the pre-clinical data obtained from animal experiments will inevitably cause a low concordance with human clinical trials, which will eventually lead to new drug development failure. Employing human induced pluripotent stem cells (iPSCs) or adult stem cells to simulate disease models can not only provide an unlimited cell materials, but also faithfully represent the genetic background of a certain disease, when iPSCs or adult stem cells derived from patients with a specific disease genetic variation are applied. In addition, gene editing methods can be used to introduce genetic variants of interest into stem cells to generate disease models. Furthermore, by establishing a cell bank with a population of iPSCs in petri dish, in vitro human genetic studies can be carried out in these cells, with GWAS and QTL studies applied to identify genetic variants that are associated with drug sensitivity or cytotoxicity. These efforts may offer valuable information for the recruitment of suitable patients for clinical trials. Therefore, stem cell-derived disease models can provide valuable resources for the pathophysiological studies of diseases as well as the drug development. In this review, we will briefly introduce the development of the liver disease models derived from stem cells and their applications in disease study and drug development.

Myosin light chain 2 marks differentiating ventricular cardiomyocytes derived from human embryonic stem cells.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have great value for studies of human cardiac development, drug discovery, disease modeling, and cell therapy. However, the mixed cardiomyocyte subtypes (ventricular-, atrial-, and nodal-like myocytes) and the maturation heterogeneity of hPSC-CMs restrain their application in vitro and in vivo. Myosin light chain 2 (MYL2, encoding the ventricular/cardiac muscle isoform MLC2v protein) is regarded as a ventricular-specific marker of cardiac myocardium; however, its restricted localization to ventricles during human heart development has been questioned. Consequently, it is currently unclear whether MYL2 definitively marks ventricular hESC-CMs. Here, by using a MYL2-Venus hESC reporter line, we characterized a time-dependent increase of the MYL2-Venus positive (MLC2v-Venus+) hESC-CMs during differentiation. We also compared the molecular, cellular, and functional properties between the MLC2v-Venus+ and MYL2-Venus negative (MLC2v-Venus-) hESC-CMs. At early differentiation stages of hESC-CMs, we reported that both MLC2v-Venus- and MLC2v-Venus+ CMs displayed ventricular-like traits but the ventricular-like cells from MLC2v-Venus+ hESC-CMs displayed more developed action potential (AP) properties than that from MLC2v-Venus- hESC-CMs. Meanwhile, about a half MLC2v-Venus- hESC-CM population displayed atrial-like AP properties, and a half showed ventricular-like AP properties, whereas only ~ 20% of the MLC2v-Venus- hESC-CMs expressed the atrial marker nuclear receptor subfamily 2 group F member 2 (NR2F2, also named as COUPTFII). At late time points, almost all MLC2v-Venus+ hESC-CMs exhibited ventricular-like AP properties. Further analysis demonstrates that the MLC2v-Venus+ hESC-CMs had enhanced Ca2+ transients upon increase of the MLC2v level during cultivation. Concomitantly, the MLC2v-Venus+ hESC-CMs showed more defined sarcomeric structures and better mitochondrial function than those in the MLC2v-Venus- hESC-CMs. Moreover, the MLC2v-Venus+ hESC-CMs were more sensitive to hypoxic stimulus than the MLC2v-Venus- hESC-CMs. These results provide new insights into the development of human ventricular myocytes and reveal a direct correlation between the expression profile of MLC2v and ventricular hESC-CM development. Our findings that MLC2v is predominantly a ventricular marker in developmentally immature hESC-CMs have implications for human development, drug screening, and disease modeling, and this marker should prove useful in overcoming issues associated with hESC-CM heterogeneity.

Hepatic miR-378 modulates serum cholesterol levels by regulating hepatic bile acid synthesis.

Rationale: An improved understanding of thyroid hormone (TH) action on cholesterol metabolism will facilitate the identification of novel therapeutic targets for hypercholesterolemia. TH-regulated microRNAs (miRNAs) have been implicated in TH-controlled biological processes; however, whether and how TH-regulated miRNAs mediate the cholesterol-lowering effect of TH remains unclear. Our aim was to identify TH-regulated microRNAs that have cholesterol-lowering effects and explore the underlying mechanism. Method: Microarray and RNA-seq were performed to identify TH-regulated microRNAs and the genes regulated by mmu-miR-378-3p (miR-378) in the liver of mice, respectively. Recombinant adenoviruses encoding miR-378, Mafg, and shRNA for Mafg, antagomiR-378, liver-specific miR-378 transgenic mice, and miR-378 knockout mice were employed to investigate the roles of hepatic miR-378 and MAFG in cholesterol and bile acid homeostasis. The levels of bile salt species were determined by using UFLC-Triple-time of flight/MS. Results: Here, we show that hepatic miR-378 is positively regulated by TH. Transient overexpression of miR-378 in the liver of mice reduces serum cholesterol levels, accompanied with an increase in the expression of key enzymes in primary bile acid synthetic pathways and corresponding increases in biliary and fecal bile acid levels. Consistently, liver-specific miR-378 transgenic mice with moderate overexpression of hepatic miR-378 display decreased serum cholesterol levels and resistance to diet-induced hypercholesterolemia, while mice lacking miR-378 exhibit defects in bile acid and cholesterol homeostasis. Mechanistically, hepatic miR-378 regulates the expression of key enzymes in both classic and alternative bile acid synthetic pathways through MAFG, a transcriptional repressor, thereby modulating bile acid and cholesterol metabolism. Conclusions: TH-responsive hepatic miR-378 is capable of modulating serum cholesterol levels by regulating both the classic and alternative BA synthetic pathways. Our study not only identifies a previously undescribed role of hepatic miR-378 but also provides new cholesterol-lowering approaches.

Optimized protocol for gene editing in adipocytes using CRISPR-Cas9 technology.

We present a detailed protocol for gene editing in adipocytes using the CRISPR-Cas technology. This protocol describes sgRNA design, preparation of lentiCRISPR-sgRNA vectors, functional validation of sgRNAs, preparation of lentiviruses, and lentiviruses transduction in adipocytes. Moreover, an optimized method of gene editing using the lentiCRISPRv2 vector expressing two sgRNAs targeting two different genes has also been described. For complete details on the use and execution of this protocol, please refer to Qiu et al. (2020).