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We performed collapsing analyses on 454,796 UK Biobank (UKB) exomes to detect gene-level associations with diabetes. Recessive carriers of nonsynonymous variants in MAP3K15 were 30% less likely to develop diabetes ( P = 5.7 × 10 −10 ) and had lower glycosylated hemoglobin (β = −0.14 SD units, P = 1.1 × 10 −24 ). These associations were independent of body mass index, suggesting protection against insulin resistance even in the setting of obesity. We replicated these findings in 96,811 Admixed Americans in the Mexico City Prospective Study ( P < 0.05)Moreover, the protective effect of MAP3K15 variants was stronger in individuals who did not carry the Latino-enriched SLC16A11 risk haplotype ( P = 6.0 × 10 −4 ). Separately, we identified a Finnish-enriched MAP3K15 protein-truncating variant associated with decreased odds of both type 1 and type 2 diabetes ( P < 0.05) in FinnGen. No adverse phenotypes were associated with protein-truncating MAP3K15 variants in the UKB, supporting this gene as a therapeutic target for diabetes.

Increased saturated fatty acid levels in membrane phospholipids have been implicated in the development of metabolic disease. Here, we tested the hypothesis that increased saturated fatty acid (SFA) content in cell membranes negatively impacts adipocyte insulin signaling. Pre-adipocyte cell models with elevated SFA levels in phospholipids were generated by disrupting the ADIPOR2 locus, which resulted in a striking two-fold increase in SFA-containing phosphatidylcholines and phosphatidylethanolamines, which persisted in differentiated adipocytes. Similar changes in phospholipid composition were observed in white adipose tissues isolated from the ADIPOR2 knockout mice. The SFA levels in phospholipids could be further increased by treating ADIPOR2-deficient cells with palmitic acid and resulted in reduced membrane fluidity and endoplasmic reticulum stress in mouse and human pre-adipocytes. Strikingly, increased SFA levels in differentiated adipocyte phospholipids had no effect on adipocyte gene expression or insulin signaling in vitro. Similarly, increased adipocyte phospholipid saturation did not impair white adipose tissue function in vivo, even in mice fed a high saturated fat diet at thermoneutrality. We conclude that increasing SFA levels in adipocyte phospholipids is well tolerated and does not affect adipocyte insulin signaling in vitro and in vivo.

AMP-activated protein kinase (AMPK) plays a key role in the cellular response to low energy stress and has emerged as an attractive therapeutic target for tackling metabolic diseases. Whilst significant progress has been made regarding the physiological role of AMPK, its function in the kidney remains only partially understood. We use a mouse model expressing a constitutively active mutant of AMPK to investigate the effect of AMPK activation on kidney function in vivo. Kidney morphology and changes in gene and protein expression were monitored and serum and urine markers were measured to assess kidney function in vivo. Global AMPK activation resulted in an early-onset polycystic kidney phenotype, featuring collecting duct cysts and compromised renal function in adult mice. Mechanistically, the cystic kidneys had increased cAMP levels and ERK activation, increased hexokinase I (Hk I) expression, glycogen accumulation and altered expression of proteins associated with autophagy. Kidney tubule-specific activation of AMPK also resulted in a polycystic phenotype, demonstrating that renal tubular AMPK activation caused the cystogenesis. Importantly, human autosomal dominant polycystic kidney disease (ADPKD) kidney sections revealed similar protein localisation patterns to that observed in the murine cystic kidneys. Our findings show that early-onset chronic AMPK activation leads to a polycystic kidney phenotype, suggesting dysregulated AMPK signalling is a contributing factor in cystogenesis.

We performed collapsing analyses on 454,796 UK Biobank (UKB) exomes to detect gene-level associations with diabetes. Recessive carriers of nonsynonymous variants in

Marginal zone (MZ) B cells are innate‐like B cells that produce polyreactive antibodies with an affinity for microbial molecular patterns and carbohydrate ligands. MZ B cells have been shown to be important in mediating immunity to various bacteria including Streptococcus pneumoniae and are also implicated in inflammatory syndromes including lupus erythematosus. The intestinal microbiota is responsible for producing short‐chain fatty acids, which can regulate immune cell function by several mechanisms including ligation of the G‐protein‐coupled receptor (GPR)43. Herein, we show that MZ B cells express Gpr43 messenger RNA and that the absence of this receptor impacts on MZ B‐cell surface marker expression and antibody production. In T‐cell‐independent responses to the hapten 4‐hydroxy‐3‐nitrophenylacetic acid (NP), mice deficient in GPR43 displayed higher serum titers of NP‐specific antibodies. Moreover, in response to a pneumococcal polysaccharide vaccine, GPR43‐deficient mice developed robust serum antibody responses and had markedly increased numbers of splenic antibody‐secreting cells, compared with control mice. Finally, serum immunoglobulin M autoantibodies to double‐stranded DNA and phosphatidylcholine were increased in resting 10–15‐week‐old mice lacking GPR43. Taken together, mice lacking GPR43 have heightened antibody responses to T‐cell‐independent antigens, which may be a result of impaired regulation of MZ B cells., Marginal zone B cells express the short‐chain fatty acid receptor GPR43. Mice lacking GPR43 have heightened responses to T‐cell‐independent antigens and have elevated levels of immunoglobulin M specific for double‐stranded DNA and phosphatidylcholine. Thus, short‐chain fatty acids may regulate marginal zone B‐cell responses to several antigens.

The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. The generation of a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene and its use in targeted ObLiGaRe results in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse allows robust and tunable genome editing granting flexibility, speed and uniformity at less cost, leading to high throughput and practical preclinical in vivo therapeutic testing., CRISPR/Cas9 technology has revolutionised the ability of scientists to make genetically modified cells and animal models. Here, the authors make a Tet-On genetically modified mouse using the Streptococcus pyogenes Cas9 and demonstrate that it can be used for generation of mice with lung cancer.

The clinical heterogeneity of heart failure has challenged our understanding of the underlying genetic mechanisms of this disease. In this respect, large-scale patient DNA sequencing studies have become an invaluable strategy for identifying potential genetic contributing factors. The complex aetiology of heart failure, however, also means that

Diabetes mellitus is a chronic health condition that can result in significant end-organ complications and is estimated to impact at least 8.5% of the global adult population. Here, we performed gene-level collapsing analysis on exome sequences from 454,796 multi-ancestry UK Biobank participants to detect genetic associations with diabetes. Rare non-synonymous variants in GCK, GIGYF1, HNF1A, and HNF4A were significantly associated (P-8) with increased risk of diabetes, whereas rare non-synonymous variants in MAP3K15 were significantly associated with reduced risk of diabetes. Recessive carriers of rare non-synonymous variants in the X chromosome gene MAP3K15 had a 30% reduced risk of diabetes (OR=0.70, 95% CI: [0.62,0.79], P=5.7×10-10), along with reduced blood glucose (beta=-0.13, 95% CI: [-0.15,-0.10], P=5.5×10-18) and reduced glycosylated haemoglobin levels (beta=-0.14, 95% CI: [-0.16,-0.11], P=1.1×10-24). Hemizygous males carrying protein-truncating variants (PTVs) in MAP3K15 demonstrated a 40% reduced risk of diabetes (OR=0.60, 95% CI: [0.45,0.81], P=0.0007). These findings were independently replicated in FinnGen, with a MAP3K15 PTV associating with decreased risk of both type 1 diabetes (T1DM) and type 2 diabetes (T2DM) (pMAP3K15 loss on diabetes was independent of body mass index, suggesting its protective effect is unlikely to be mediated via the insulin resistance pathway. Tissue expression profile of MAP3K15 indicates a possible involvement of pancreatic islet cell or stress response pathways. No safety concerns were identified among heterozygous or recessive MAP3K15 PTV carriers across over 15,719 studied endpoints in the UK Biobank. Human population genetic evidence supports MAP3K15 inhibition as a novel therapeutic target for diabetes.

Cells and organisms require proper membrane composition to function and develop. Phospholipids are the major component of membranes and are primarily acquired through the diet. Given great variability in diet composition, cells must be able to deploy mechanisms that correct deviations from optimal membrane composition and properties. Here, using unbiased lipidomics and proteomics, we found that the embryonic lethality in mice lacking the fluidity regulators Adiponectin Receptors 1 and 2 (AdipoR1/2) is associated with aberrant high saturation of the membrane phospholipids. Using mouse embryonic fibroblasts (MEFs) derived from AdipoR1/2-KO embryos, human cell lines and the model organism C. elegans we found that, mechanistically, AdipoR1/2-derived sphingosine 1-phosphate (S1P) signals in parallel through S1PR3-SREBP1 and PPAR{gamma} to sustain the expression of the fatty acid desaturase SCD and maintain membrane properties. Thus, our work identifies an evolutionary conserved pathway by which cells and organism maintain membrane homeostasis and adapt to a variable environment. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/456080v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@b6730aorg.highwire.dtl.DTLVardef@3a95e3org.highwire.dtl.DTLVardef@469027org.highwire.dtl.DTLVardef@a21790_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefCells and organisms require proper membrane composition to function and develop. Ruiz et al. identified AdipoR1/2-derived S1P as essential for membrane homeostasis in mammals and invertebrates. S1P signals through SREBP and PPAR{gamma} to sustain fatty acid desaturation. Targeting AdipoR1/2 pathway may be a therapeutic approach for disorders with dysfunctional membranes. Highlights- Excess membrane phospholipid saturation explains AdipoR1/2 DKO embryonic lethality - AdipoR1/2 DKO membrane defects are rescued by S1P - Parallel activation of S1PR3 and PPAR{gamma} by S1P mediates AdipoR2 membrane protection - Fatty acid desaturation is the final outputs of the AdipoR2-S1P signaling pathway

The clinical heterogeneity of heart failure has challenged our understanding of the underlying genetic mechanisms of this disease. In this respect, large-scale patient DNA sequencing studies have become an invaluable strategy for identifying potential genetic contributing factors. The complex aetiology of heart failure, however, also means that in vivo models are vital to understand the links between genetic perturbations and functional impacts. Traditional approaches (e.g. genetically-modified mice) are optimal for assessing small numbers of proposed target genes, but less practical when multiple targets are identified. The zebrafish, in contrast, offers great potential for higher throughput in vivo gene functional assessment to aid target prioritisation and support definitive studies undertaken in mice. Here we used whole-exome sequencing and bioinformatics on human patient data to identify 3 genes (API5, HSPB7, and LMO2) suggestively associated with heart failure that were also predicted to play a broader role in disease aetiology. The role of these genes in cardiovascular system development and function was then further investigated using in vivo CRISPR/Cas9-mediated gene mutation analysis in zebrafish. We observed multiple impacts in F0 knockout zebrafish embryos (crispants) following effective somatic mutation, including reductions in ventricle size, pericardial oedema, and chamber malformation. In the case of lmo2, there was also a significant impact on cardiovascular function as well as an expected reduction in erythropoiesis. The data generated from both the human in silico and zebrafish in vivo assessments undertaken supports roles for API5, HSPB7, and LMO2 in human cardiovascular disease and identifies them as potential drug targets for further investigation. The data presented also supports the use of human in silico genetic variant analysis, in combination with zebrafish crispant phenotyping, as a powerful approach for assessing gene function as part of an integrated multi-level drug target validation strategy.

Recent advances in induced pluripotent stem cells (iPSCs), genome editing technologies and 3D organoid model systems highlight opportunities to develop new in vitro human disease models to serve drug discovery programs. An ideal disease model would accurately recapitulate the relevant disease phenotype and provide a scalable platform for drug and genetic screening studies. Kidney organoids offer a high cellular complexity that may provide greater insights than conventional single-cell type cell culture models. However, genetic manipulation of the kidney organoids requires prior generation of genetically modified clonal lines, which is a time and labor consuming procedure. Here, we present a methodology for direct differentiation of the CRISPR-targeted cell pools, using a doxycycline-inducible Cas9 expressing hiPSC line for high efficiency editing to eliminate the laborious clonal line generation steps. We demonstrate the versatile use of genetically engineered kidney organoids by targeting the autosomal dominant polycystic kidney disease (ADPKD) genes: PKD1 and PKD2. Direct differentiation of the respective knockout pool populations into kidney organoids resulted in the formation of cyst-like structures in the tubular compartment. Our findings demonstrated that we can achieve > 80% editing efficiency in the iPSC pool population which resulted in a reliable 3D organoid model of ADPKD. The described methodology may provide a platform for rapid target validation in the context of disease modeling.

Direct in vivo reprogramming of cardiac fibroblasts into myocytes is an attractive therapeutic intervention in resolving myogenic deterioration. Current transgene-dependent approaches can restore cardiac function, but dependence on retroviral delivery and persistent retention of transgenic sequences are significant therapeutic hurdles. Chemical reprogramming has been established as a legitimate method to generate functional cell types, including those of the cardiac lineage. Here, we have extended this approach to generate progenitor cells that can differentiate into endothelial cells and cardiomyocytes using a single inhibitor protocol. Depletion of terminally differentiated cells and enrichment for proliferative cells result in a second expandable progenitor population that can robustly give rise to myofibroblasts and smooth muscle. Deployment of a genome-wide knockout screen with clustered regularly interspaced short palindromic repeats-guide RNA library to identify novel mediators that regulate the reprogramming revealed the involvement of DNA methyltransferase 1-associated protein 1 (Dmap1). Loss of Dmap1 reduced promoter methylation, increased the expression of Nkx2-5, and enhanced the retention of self-renewal, although further differentiation is inhibited because of the sustained expression of Cdh1. Our results hence establish Dmap1 as a modulator of cardiac reprogramming and myocytic induction. Stem Cells 2019;37:958–972

Background Plasma concentration of low-density lipoprotein (LDL) cholesterol is a well-established risk factor for cardiovascular disease. Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9), which regulates cholesterol homeostasis, has recently emerged as an approach to reduce cholesterol levels. The development of humanized animal models is an important step to validate and study human drug targets, and use of genome and base editing has been proposed as a mean to target disease alleles. Results To address the lack of validated models to test the safety and efficacy of techniques to target human PCSK9, we generated a liver-specific human PCSK9 knock-in mouse model (hPCSK9-KI). We showed that plasma concentrations of total cholesterol were higher in hPCSK9-KI than in wildtype mice and increased with age. Treatment with evolocumab, a monoclonal antibody that targets human PCSK9, reduced cholesterol levels in hPCSK9-KI but not in wildtype mice, showing that the hypercholesterolemic phenotype was driven by overexpression of human PCSK9. CRISPR-Cas9-mediated genome editing of human PCSK9 reduced plasma levels of human and not mouse PCSK9, and in parallel reduced plasma concentrations of total cholesterol; genome editing of mouse Pcsk9 did not reduce cholesterol levels. Base editing using a guide RNA that targeted human and mouse PCSK9 reduced plasma levels of human and mouse PCSK9 and total cholesterol. In our mouse model, base editing was more precise than genome editing, and no off-target editing nor chromosomal translocations were identified. Conclusions Here, we describe a humanized mouse model with liver-specific expression of human PCSK9 and a human-like hypercholesterolemia phenotype, and demonstrate that this mouse can be used to evaluate antibody and gene editing-based (genome and base editing) therapies to modulate the expression of human PCSK9 and reduce cholesterol levels. We predict that this mouse model will be used in the future to understand the efficacy and safety of novel therapeutic approaches for hypercholesterolemia. Electronic supplementary material The online version of this article (10.1186/s12915-018-0624-2) contains supplementary material, which is available to authorized users.

Introduction: While the vast majority of research investigating the role of ghrelin or its receptor, GHS-R1a, in growth, feeding, and metabolism has been conducted in male rodents, very little is known about sex differences in this system. Furthermore, the role of GHS-R1a signaling in the control of pulsatile GH secretion and its link with growth or metabolic parameters has never been characterized. Methods: We assessed the sex-specific contribution of GHS-R1a signaling in the activity of the GH/IGF-1 axis, metabolic parameters, and feeding behavior in adolescent (5–6 weeks old) or adult (10–19 weeks old) GHS-R KO (Ghsr−/−) and WT (Ghsr+/+) male and female mice. Results: Adult Ghsr−/− male and female mice displayed deficits in weight and linear growth that were correlated with reduced GH pituitary contents in males only. GHS-R1a deletion was associated with reduced meal frequency and increased meal intervals, as well as reduced hypothalamic GHRH and NPY mRNA in males, not females. In adult, GH release from Ghsr−/− mice pituitary explants ex vivo was reduced independently of the sex. However, in vivo pulsatile GH secretion decreased in adult but not adolescent Ghsr−/− females, while in males, GHS-R1a deletion was associated with reduction in pulsatile GH secretion during adolescence exclusively. In males, linear growth did not correlate with pulsatile GH secretion, but rather with ApEn, a measure that reflects irregularity of the rhythmic secretion. Fat mass, plasma leptin concentrations, or ambulatory activity did not predict differences in GH secretion. Discussion/Conclusion: These results point to a sex-dependent dimorphic effect of GHS-R1a signaling to modulate pulsatile GH secretion and meal pattern in mice with different compensatory mechanisms occurring in the hypothalamus of adult males and females after GHS-R1a deletion. Altogether, we show that GHS-R1a signaling plays a more critical role in the regulation of pulsatile GH secretion during adolescence in males and adulthood in females.

Prokaryotic restriction enzymes, recombinases and Cas proteins are powerful DNA engineering and genome editing tools. However, in many primary cell types, the efficiency of genome editing remains low, impeding the development of gene- and cell-based therapeutic applications. A safe strategy for robust and efficient enrichment of precisely genetically engineered cells is urgently required. Here, we screen for mutations in the receptor for Diphtheria Toxin (DT) which protect human cells from DT. Selection for cells with an edited DT receptor variant enriches for simultaneously introduced, precisely targeted gene modifications at a second independent locus, such as nucleotide substitutions and DNA insertions. Our method enables the rapid generation of a homogenous cell population with bi-allelic integration of a DNA cassette at the selection locus, without clonal isolation. Toxin-based selection works in both cancer-transformed and non-transformed cells, including human induced pluripotent stem cells and human primary T-lymphocytes, as well as it is applicable also in vivo, in mice with humanized liver. This work represents a flexible, precise, and efficient selection strategy to engineer cells using CRISPR-Cas and base editing systems., Genome engineering in cell lines or human stem cells often has poor efficiency, limiting the development of research and therapeutic applications. Here, the authors use a toxin-based selection system for precise bi-allelic engineering in cells and in vivo.

The generation of tissue-specific integrated networks (INs) through the merging of transcriptional regulatory, protein-protein interaction and genome-scale metabolic networks allows for a rigorous investigation of the biological processes that are altered in cardiovascular disease (CVD) subjects. We developed INs for left ventricle (LV) and visceral adipose tissue (omentum) of healthy subjects and compared these with LV and epicardial fat INs for CVD subjects. We investigated changes in co-regulation for enzymes in pathways that are known to be associated with the development and progression of CVD for each tissue type. Global gene expression changes between healthy and CVD subjects were determined for both tissue types and reporter metabolite analyses were performed. The application and combination of these different network approaches enabled us to associate low levels of Coenzyme Q10 with cardiac remodelling in the LV. We also found increasing homocysteine levels with dysregulation of the adipocytokine pathway in the epicardial fat of CVD subjects and validated our predictions by detecting increased plasma metabolite levels associated with homocysteine in CVD subjects.

Aging, obesity, and insulin resistance are associated with low levels of PGC1 alpha and PGC1 beta coactivators and defective mitochondrial function. We studied mice deficient for PGC1 alpha and PGC1 beta [double heterozygous (DH)] to investigate their combined pathogenic contribution. Contrary to our hypothesis, DH mice were leaner, had increased energy dissipation, a pro-thermogenic profile in BAT and WAT, and improved carbohydrate metabolism compared to wild types. WAT showed upregulation of mitochondriogenesis/oxphos machinery upon allelic compensation of PGC1 alpha 4 from the remaining allele. However, DH mice had decreased mitochondrial OXPHOS and biogenesis transcriptomes in mitochondria-rich organs. Despite being metabolically healthy, mitochondrial defects in DH mice impaired muscle fiber remodeling and caused qualitative changes in the hepatic lipidome. Our data evidence first the existence of organ-specific compensatory allostatic mechanisms are robust enough to drive an unexpected phenotype. Second, optimization of adipose tissue bioenergetics is sufficient to maintain a healthy metabolic phenotype despite a broad severe mitochondrial dysfunction in other relevant metabolic organs. Third, the decrease in PGC1s in adipose tissue of obese and diabetic patients is in contrast with the robustness of the compensatory upregulation in the adipose of the DH mice. EC | FP7 | FP7 Health (HEALTH), Grant/Award Number: [HEALTH-F4-2008-223450; RCUK | Medical Research Council (MRC), Grant/Award Number: MC_UU_12012/2 and MC_UU_00014/5; European Commission (EC), Grant/ Award Number: MEIF-CT-2005-023061; Wellcome Trust (Wellcome), Grant/Award Number: 208363/Z/17/Z

How cells maintain vital membrane lipid homeostasis while obtaining most of their constituent fatty acids from a varied diet remains largely unknown. Here, we used transcriptomics, lipidomics, growth and respiration assays, and membrane property analyses in human HEK293 cells or human umbilical vein endothelial cells (HUVEC) to show that the function of AdipoR2 is to respond to membrane rigidification by regulating many lipid metabolism genes. We also show that AdipoR2-dependent membrane homeostasis is critical for growth and respiration in cells challenged with saturated fatty acids. Additionally, we found that AdipoR2 deficiency causes transcriptome and cell physiological defects similar to those observed in SREBP-deficient cells upon SFA challenge. Finally, we compared several genes considered important for lipid homeostasis, namely AdipoR2, SCD, FADS2, PEMT and ACSL4, and found that AdipoR2 and SCD are the most important among these to prevent membrane rigidification and excess saturation when human cells are challenged with exogenous SFAs. We conclude that AdipoR2-dependent membrane homeostasis is one of the primary mechanisms that protects against exogenous SFAs.

Background and Aims: The proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in cholesterol homeostasis, and its inhibition represents an effective therapy to lower low-density lipoprotein cholesterol (LDL-C) levels. In this study, we examined the impact of the PCSK9 rs11591147 loss-of-function (LOF) variant on liver damage in a multicenter collection of patients at risk of nonalcoholic steatohepatitis (NASH), in clinical samples and experimental models. Methods: We considered 1874 consecutive individuals at risk of NASH as determined by histology. The SNP rs11591147, encoding for the p.R46L variant of PCSK9, was genotyped by TaqMan assays. We also evaluated 1) PCSK9 mRNA hepatic expression in human liver, and 2) the impact of a NASH-inducing diet in mice with hepatic overexpression of human PCSK9. Results: Carriers of PCSK9 rs11591147 had lower circulating LDL-C levels and were protected against nonalcoholic fatty liver disease (NAFLD) (OR: 0.42; 95% CI: 0.22-0.81; P=.01), NASH (OR: 0.48; 95% CI: 0.26-0.87; P=.01) and more severe fibrosis (OR: 0.55; 95% CI: 0.32-0.94; P=.03) independently of clinical, metabolic and genetic confounding factors. PCSK9 hepatic expression was directly correlated with liver steatosis (P=.03). Finally, liver-specific overexpression of human PCSK9 in male mice drives NAFLD and fibrosis upon a dietary challenge. Conclusions: In individuals at risk of NASH, PCSK9 was induced with hepatic fat accumulation and PCSK9 rs11591147 LOF variant was protective against liver steatosis, NASH and fibrosis, suggesting that PCSK9 inhibition may be a new therapeutic strategy to treat NASH.

Membrane lipid composition influences vital processes in all types of cells. The mechanisms by which cells maintain membrane lipid homeostasis while obtaining most of their constituent fatty acids from a varied diet remain largely unknown. In an attempt to discover such mechanisms, we performed an unbiased forward genetic screen inCaenorhabditis elegansand conclude that the adiponectin receptor 2 (AdipoR2) pathway is essential to prevent saturated fat-mediated cellular toxicity. Transcriptomics, lipidomics and membrane property analyses in human HEK293 cells and primary human endothelial cells further support our conclusion that the essential function of AdipoR2 is to respond to membrane rigidification by promoting fatty acid desaturation. Our results demonstrate that AdipoR2-dependent regulation of membrane homeostasis is a fundamental mechanism conserved from nematodes to mammals that prevents saturated fat-mediated lipotoxicity.ONE SENTENCE SUMMARYThe AdipoR2 protein insures membrane homeostasis in response to dietary saturated fatty acids that promote membrane rigidification.

The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. We have generated a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene. Targeted ObLiGaRe resulted in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse can be utilized for robust and tunable genome editing allowing for flexibility, speed and uniformity at reduced cost, leading to high throughput and practical preclinical in vivo therapeutic testing.

Background and Aims: The proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in cholesterol homeostasis, and its inhibition represents an effective therapy to lower LDL-C levels. In this study, we examined the impact of PCSK9 rs11591147 loss-of-function (LOF) variant on liver damage in a multicenter collection of patients at risk of nonalcoholic steatohepatitis (NASH), in clinical samples and experimental models. Methods: We considered 1,874 consecutive individuals at risk of NASH as determined by histology. The SNP rs11591147, encoding for the p.R46L variant of PCSK9, was genotyped by TaqMan assays. We also evaluated 1) PCSK9 mRNA hepatic expression in human liver, and 2) the impact of a NASH-inducing diet in mice with hepatic overexpression of human PCSK9. Results: Carriers of PCSK9 rs11591147 had lower circulating LDL-C levels and were protected against NAFLD (OR 0.42;95%C.I0.22-0.81;P=0.01), NASH (OR 0.39;95%C.I.0.20-0.75;P=0.005) and more severe fibrosis (OR 0.44;95%C.I.0.26-0.74;P=0.002) independently of clinical, metabolic and genetic confounding factors. PCSK9 hepatic expression was directly correlated with steatosis (P=0.03). Finally, liver-specific overexpression of human PCSK9 in mice drives NAFLD and fibrosis upon a dietary challenge. Conclusions: In individuals at risk, PCSK9 was induced with hepatic fat accumulation and PCSK9 rs11591147 LOF variant was protective against liver steatosis, NASH and fibrosis, suggesting PCSK9 inhibition may be a new therapeutic strategy to treat NASH. Funding Statement: This work was supported by project grant by the Swedish Research Council [Vetenskapsradet (VR), 2016-01527], the Swedish state under the Agreement between the Swedish government and the county councils (the ALF-agreement) [SU 2018-04276], the Novonordisk Foundation Grant for Excellence in Endocrinology [Excellence Project, 9321- 430], the Swedish Diabetes Foundation [DIA 2017-205], the Swedish Heart Lung Foundation [20120533], the Wallenberg Academy Fellows from the Knut and Alice Wallenberg Foundation [KAW 2017.0203]. LV was supported by MyFirst Grant AIRC n.16888, Ricerca Finalizzata Ministero della Salute [RF-2016-02364358], Ricerca corrente Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Fondazione Sviluppo Ca Granda [PR-0316], the European Union Programme Horizon 2020 [No. 777377]. Declaration of Interests: SR has been consulting for AMGEN, SANOFI, Astra Zeneca, Pfizer, AKCEA, Celgene, Medacorp, Foresite Labs, CAMP4 and received research grants from Astra Zeneca, AMGEN, SANOFI. SB, DK-P, A-CA, GP, MB-Y and DL are AstraZeneca employees. LV reports having received during the last 5 years speaking fees from MSD, Gilead, AlfaSigma, AbbVie, having served as a consultant of: Gilead, Pfizer, Astra Zeneca, Novo Nordisk, Diatech Pharmacogenetics and Intercept, and having received research grants from: Gilead. Ethics Approval Statement: The study was carried out in accordance with the principles of the Helsinki Declaration, and with local and national laws. Approval was obtained from Internal Review Boards and their Ethics Committees, and written informed consent for the study was obtained from all patients All animal experiments were performed with humane care and were approved by the Gothenburg Ethics Committee for Experimental Animals in Sweden. The holding facility has received full accreditation from the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

CRISPR–Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications1–6 but identifying unwanted off-target mutations is important for clinical translation7. A well-validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe ‘verification of in vivo off-targets’ (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR–Cas nucleases in vivo. We use VIVO and a guide RNA deliberately designed to be promiscuous to show that CRISPR–Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVO to show that appropriately designed guide RNAs can direct efficient in vivo editing in mouse livers with no detectable off-target mutations. VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing. A strategy developed to define off-target effects of gene-editing nucleases in whole organisms is validated and leveraged to show that CRISPR–Cas9 nucleases can be used effectively in vivo without inducing detectable off-target mutations.