Enhancing the genome editing toolbox: genome wide CRISPR arrayed libraries.

CRISPR-Cas9 technology has accelerated biological research becoming routine for many laboratories. It is rapidly replacing conventional gene editing techniques and has high utility for both genome-wide and gene-focussed applications. Here we present the first individually cloned CRISPR-Cas9 genome wide arrayed sgRNA libraries covering 17,166 human and 20,430 mouse genes at a complexity of 34,332 sgRNAs for human and 40,860 sgRNAs for the mouse genome. For flexibility in generating stable cell lines the sgRNAs have been cloned in a lentivirus backbone containing PiggyBac transposase recognition elements together with fluorescent and drug selection markers. Over 95% of tested sgRNA induced specific DNA cleavage as measured by CEL-1 assays. Furthermore, sgRNA targeting GPI anchor protein pathway genes induced loss of function mutations in human and mouse cell lines measured by FLAER labelling. These arrayed libraries offer the prospect for performing screens on individual genes, combinations as well as larger gene sets. They also facilitate rapid deconvolution of signals from genome-wide screens. This set of vectors provide an organized comprehensive gene editing toolbox of considerable scientific value.

High-efficiency genome editing via 2A-coupled co-expression of fluorescent proteins and zinc finger nucleases or CRISPR/Cas9 nickase pairs.

Targeted endonucleases including zinc finger nucleases (ZFNs) and clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas9 are increasingly being used for genome editing in higher species. We therefore devised a broadly applicable and versatile method for increasing editing efficiencies by these tools. Briefly, 2A peptide-coupled co-expression of fluorescent protein and nuclease was combined with fluorescence-activated cell sorting (FACS) to allow for efficient isolation of cell populations with increasingly higher nuclease expression levels, which translated into increasingly higher genome editing rates. For ZFNs, this approach, combined with delivery of donors as single-stranded oligodeoxynucleotides and nucleases as messenger ribonucleic acid, enabled high knockin efficiencies in demanding applications, including biallelic codon conversion frequencies reaching 30-70% at high transfection efficiencies and ∼ 2% at low transfection efficiencies, simultaneous homozygous knockin mutation of two genes with ∼ 1.5% efficiency as well as generation of cell pools with almost complete codon conversion via three consecutive targeting and FACS events. Observed off-target effects were minimal, and when occurring, our data suggest that they may be counteracted by selecting intermediate nuclease levels where off-target mutagenesis is low, but on-target mutagenesis remains relatively high. The method was also applicable to the CRISPR/Cas9 system, including CRISPR/Cas9 mutant nickase pairs, which exhibit low off-target mutagenesis compared to wild-type Cas9.

Curcumin eliminates the inhibitory effect of advanced glycation end-products (AGEs) on gene expression of AGE receptor-1 in hepatic stellate cells in vitro.

Diabetes is featured by hyperglycemia, which facilitates the formation of advanced glycation end-products (AGEs). AGEs are a causal factor in development of diabetic complications. AGE receptor-1 (AGE-R1) is responsible for detoxification and clearance of AGEs. Type 2 diabetes mellitus is commonly accompanied by non-alcoholic steatohepatitis, which could cause hepatic fibrosis. Little attention has been paid to effects of AGEs on hepatic fibrogenesis. Curcumin, a phytochemical from turmeric, has been reported to inhibit the activation of hepatic stellate cells (HSCs), the major effectors during hepatic fibrogenesis, and to protect against hepatic fibrogenesis in vitro and in vivo. The current study was designed to evaluate the effects of AGEs on inducing HSC activation, to assess the role of curcumin in diminishing the AGE effects, and to explore the underlying mechanisms. Our results showed that AGEs stimulated HSC activation by inducing cell proliferation and expression of genes relevant to HSC activation, which were abrogated by curcumin. Curcumin induced gene expression of AGE-R1 in passaged HSCs, which might facilitate the attenuation of the stimulatory effects of AGEs on the activation of HSCs. Further experiments revealed that curcumin inhibited the activity of extracellular signal-regulated kinase (ERK), and induced gene expression and the activity of peroxisome proliferator-activated receptor-gamma (PPARγ), leading to the induction of the AGE-R1 gene expression. In summary, AGEs stimulated HSC activation. Curcumin eliminated the AGE effects at least partially by inducing the AGE-R1 gene expression. The process was mediated by inhibiting ERK activity, inducing gene expression of PPARγ and stimulating its transactivity.

Curcumin inhibits gene expression of receptor for advanced glycation end-products (RAGE) in hepatic stellate cells in vitro by elevating PPARγ activity and attenuating oxidative stress.

BACKGROUND AND PURPOSE: Diabetes is characterized by hyperglycaemia, which facilitates the formation of advanced glycation end-products (AGEs). Type 2 diabetes mellitus is commonly accompanied by non-alcoholic steatohepatitis, which could lead to hepatic fibrosis. Receptor for AGEs (RAGE) mediates effects of AGEs and is associated with increased oxidative stress, cell growth and inflammation. The phytochemical curcumin inhibits the activation of hepatic stellate cells (HSCs), the major effectors during hepatic fibrogenesis. The aim of this study was to explore the underlying mechanisms of curcumin in the elimination of the stimulating effects of AGEs on the activation of HSCs. We hypothesize that curcumin eliminates the effects of AGEs by suppressing gene expression of RAGE. EXPERIMENTAL APPROACH: Gene promoter activities were evaluated by transient transfection assays. The expression of rage was silenced by short hairpin RNA. Gene expression was analysed by real-time PCR and Western blots. Oxidative stress was evaluated. KEY RESULTS: AGEs induced rage expression in cultured HSCs, which played a critical role in the AGEs-induced activation of HSCs. Curcumin at 20 µM eliminated the AGE effects, which required the activation of PPARγ. In addition, curcumin attenuated AGEs-induced oxidative stress in HSCs by elevating the activity of glutamate-cysteine ligase and by stimulating de novo synthesis of glutathione, leading to the suppression of gene expression of RAGE. CONCLUSION AND IMPLICATIONS: Curcumin suppressed gene expression of RAGE by elevating the activity of PPARγ and attenuating oxidative stress, leading to the elimination of the AGE effects on the activation of HSCs. LINKED ARTICLE: This article is commented on by Stefanska, pp. 2209-2211 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.01959.x.

Curcumin inhibits srebp-2 expression in activated hepatic stellate cells in vitro by reducing the activity of specificity protein-1.

Elevated levels of cholesterol/low-density lipoprotein (LDL) are a risk factor for the development of nonalcoholic steatohepatitis and its associated hepatic fibrosis. However, underlying mechanisms remain elusive. We previously reported that curcumin induced gene expression of peroxisome proliferator-activated receptor (PPAR)-gamma and stimulated its activity, leading to the inhibition of the activation of hepatic stellate cells (HSCs), the major effector cells during hepatic fibrogenesis. We recently showed that curcumin suppressed gene expression of LDL receptor in activated HSCs in vitro by repressing gene expression of the transcription factor sterol regulatory element binding protein-2 (SREBP-2), leading to the reduction in the level of intracellular cholesterol in HSCs and to the attenuation of the stimulatory effects of LDL on HSCs activation. The current study aimed at exploring molecular mechanisms by which curcumin inhibits srebp-2 expression in HSCs. Promoter deletion assays, mutagenesis assays, and EMSAs localize a specificity protein-1 (SP-1) binding GC-box in the srebp-2 promoter, which is responsible for enhancing the promoter activity and responding to curcumin in HSCs. Curcumin suppresses gene expression of SP-1 and reduces its trans-activation activity, which are mediated by the activation of PPARgamma. The inhibitory effect of curcumin on SP-1 binding to the GC-box is confirmed by chromatin immuno-precipitation. In summary, our results demonstrate that curcumin inhibits srebp-2 expression in cultured HSCs by activating PPARgamma and reducing the SP-1 activity, leading to the repression of ldlr expression. These results provide novel insights into molecular mechanisms by which curcumin inhibits LDL-induced HSC activation.

Curcumin eliminates oxidized LDL roles in activating hepatic stellate cells by suppressing gene expression of lectin-like oxidized LDL receptor-1.

Type II diabetes mellitus (T2DM) is often accompanied by non-alcoholic steatohepatitis (NASH) and associated with hypercholesterolemia, that is, increased levels of plasma low-density lipoprotein (LDL) and oxidized LDL (ox-LDL). Approximately one-third of NASH develops hepatic fibrosis. The role of hypercholesterolemia in T2DM and NASH-associated hepatic fibrogenesis remains obscure. We previously reported that the phytochemical curcumin inhibited the activation of hepatic stellate cells (HSCs), the major effector cells during hepatic fibrogenesis, and protected the liver from fibrogenesis in vitro and in vivo. The aims of this study are to evaluate the role of ox-LDL in activation of HSCs, to assess curcumin effects on eliminating the role of ox-LDL, and to further explore the underlying mechanisms. In this report, we observe that ox-LDL alters the expression of genes closely relevant to HSC activation, which is eliminated by curcumin. Curcumin suppresses gene expression of lectin-like oxidized LDL receptor-1 (LOX-1), leading to the blockade of the transport of extracellular ox-LDL into cells. This suppressive effect of curcumin results from the interruption of Wnt signaling and the activation of peroxisome proliferator-activated receptor-gamma (PPARgamma). In conclusion, these results support our initial hypothesis and demonstrate that ox-LDL stimulates HSC activation, which is eliminated by curcumin by suppressing lox-1 expression by interrupting Wnt signaling and stimulating PPARgamma activity. These results provide novel insights into the role of ox-LDL in T2DM and NASH-associated hepatic fibrogenesis and mechanisms by which curcumin suppresses ox-LDL-induced HSC activation, as well as the implication of curcumin in the treatment of T2DM and NASH-associated hepatic fibrosis.

Curcumin suppresses expression of low-density lipoprotein (LDL) receptor, leading to the inhibition of LDL-induced activation of hepatic stellate cells.

BACKGROUND AND PURPOSE: Obesity is often accompanied by hypercholesterolemia characterized by elevated levels of plasma low-density lipoprotein (LDL) and associated with non-alcoholic steatohepatitis, which could progress to hepatic fibrosis. Hepatic stellate cells (HSCs) are the major effectors of hepatic fibrogenesis. This study aims to clarify effects of LDL on activation of HSC, to evaluate roles of curcumin in suppressing these effects and to further elucidate the underlying molecular mechanisms. EXPERIMENTAL APPROACHES: HSCs were prepared from rats and cell proliferation was measured by cell proliferation assays (MTS assays). Transient transfection assays were performed to evaluate gene promoter activities. Real-time polymerase chain reaction and Western blotting were used to analyse the expression of genes. KEY RESULTS: LDL stimulated HSC activation in vitro, which was attenuated by curcumin. Curcumin reduced the abundance of LDL receptor (LDLR) in activated HSCs, decreasing cellular cholesterol. Curcumin-dependent activation of peroxisome proliferator-activated receptor-gamma (PPARgamma) differentially regulated the expression of the transcription factors, sterol regulatory element-binding proteins (SREBPs), in activated HSCs, resulting in the suppression of LDLR gene expression. CONCLUSIONS AND IMPLICATIONS: Curcumin suppressed LDLR gene expression in activated HSCs in vitro by activating PPARgamma and differentially regulating gene expression of SREBPs, reducing cellular cholesterol and attenuating the stimulatory effects of LDL on HSC activation. These results provide novel insights into the roles and mechanisms of curcumin in the inhibition of LDL-induced HSC activation. This curcumin, a constituent of turmeric, may be useful in preventing hypercholesterolemia-associated hepatic fibrogenesis.

Constitutive coactivator of peroxisome proliferator-activated receptor (PPARgamma), a novel coactivator of PPARgamma that promotes adipogenesis.

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays essential roles in adipogenesis by transcriptionally regulating adipocyte-specific genes through recruitment of coregulators including coactivators and corepressors. However, the precise repertoire of coactivators required for PPARgamma transactivation remains unresolved. In this report, we cloned and characterized a novel PPARgamma interacting protein, constitutive coactivator of PPARgamma (CCPG), which is expressed in multiple adult tissues and throughout embryonic development. CCPG is localized in nucleus and contains four LXXLL motifs, which are characteristic for nuclear receptor coactivators. A delineation of CCPG-PPARgamma interaction by glutathione-S-transferase pull-down and coimmunoprecipitation assays indicated that CCPG interacts with the hinge region of PPARgamma in a ligand-independent manner. However, mutation of four motifs of LXXLL to LXXAA in CCPG does not compromise its interaction with PPARgamma, suggesting LXXLL motif is not required for the interaction. Glutathione-S-transferase pull-down assays showed that CCPG binds to retinoic X receptor-alpha and estrogen receptor-alpha independent of their ligands, but not to thyroid hormone receptor-beta. CCPG coactivates PPARgamma in PPAR response element reporter assays, and the N terminus (amino acids 1-561) of CCPG acts to significantly augment the transactivation of PPARgamma, whereas the C terminus (amino acids 562-786) represses PPARgamma activity, indicating the N terminus possesses the activation domain. Using an adenoviral-mediated system, we also revealed that overexpression of CCPG promoted differentiation of OP9 preadipocyte into adipocyte, and knockdown of CCPG by RNA interference blocked this process, as examined by Oil Red O staining and Western blots of adipocyte-specific protein, adiponectin, and perilipin. Taken together, our data indicate that CCPG is a bona fide coactivator and promotes adipogenesis in a PPARgamma-dependent manner.

Upregulation of hypoxia-induced mitogenic factor in bacterial lipopolysaccharide-induced acute lung injury.

Hypoxia-induced mitogenic factor (HIMF), also known as FIZZ1 (found in inflammatory zone), plays important roles in lung inflammation. We found that intraperitoneal injection of lipopolysaccharide (LPS) induced intensive HIMF production exclusively in mouse lung, but not in the heart, liver, spleen or kidney. This HIMF production, at least partly, contributes to LPS-induced vascular cell adhesion molecule-1 (VCAM-1) upregulation and mononuclear cell sequestration to lung parenchyma, while protecting alveolar type II cells from LPS-resulted decrease in surfactant protein-C production and cell death. These data indicate that HIMF participates in LPS-induced acute lung injury and inflammation through modulating VCAM-1 and SP-C expression.

[Affection of metallothionein-3 to dUTPase's accommodating cellular toxicity of dUTP].

Metallothionein-3 (MT-3), renamed as growth inhibitory factor (GIF), is a brain specific member of the metallothionein family. Human dUTPase is a recently found protein in brain that can interact with hMT-3. They have the growth inhibitory activity on neuron cell by interaction. To study the affection of hMT-3 to dUTPase's eliminating the cellular toxicity caused by dUTP, the pSVHA-dUTPase and pFLag-hMT-3 genes have been transfected into HEK293 cells. In addition, the dUTPase and hMT-3 proteins were expressed in BL21 to study the role of hMT-3 on the hydrolyzation of dUTP by dUTPase. The results demonstrate that the cells co-transfected with dUTPase and hMT-3 genes have more strong resistibility to dUTP than the cells transfected only with dUTPase gene. And that the hMT-3 protein can accelerate the hydrolyzation of dUTP by dUTPase. All these indicate that hMT-3 can cooperate with dUTPase to protect better the 293 cells from dUTP. This research offered the theoretic elements for the application of hMT-3 and dUTPase in chemic cure.